Search for notes by fellow students, in your own course and all over the country.
Browse our notes for titles which look like what you need, you can preview any of the notes via a sample of the contents. After you're happy these are the notes you're after simply pop them into your shopping cart.
Document Preview
Extracts from the notes are below, to see the PDF you'll receive please use the links above
Nucleic Acid Isolation and
Purification
th
4 edition
Sea Otters
Sea otters are the smallest marine mammals and are adapted to spending almost all of their
lives in the ocean
...
They belong to the species „Enhydria lutris“,
from the Greek „en hydra“, meaning „in the water“, and the Latin „lutris“, meaning otter
...
They prefer
coastal waters and only swim out into the deep ocean when travelling between islands and
the mainland
...
Instead, sea otters depend on their fur to
keep warm
...
Any water needs to be squeezed out and air needs
to be blown into the fur
...
That‘s why cleaning is essential
...
In addition to their thick coats, sea otters have another defense against the cold ocean water
...
As a result, sea otters need to eat a lot, sometimes as much as 25% of their body weight each
day
...
As they must constantly eat to survive, otters spend a lot of
time diving for food
...
Capturing their prey is not always an easy task
...
Smaller shellfish clinging to rocks can be prised off by the otter’s strong arms
...
Using rocks as hammers, the otters are capable of dislodging the abalone with
several firm strikes from the side
...
Otters can dig with their
forepaws, like dogs, and sometimes use rocks as digging implements
...
Once the otter has found its food, it carries it to the surface to eat
...
Although sea otters have strong teeth that can bite through crab shells
and some shellfish, other sources of prey, e
...
abalones, have to be cracked open first
...
Some otters place a big rock on their chest and then smash their prey on the rock until the
shell breaks open
...
Some otters use numerous short, rapid strikes, while
others administer a few strong blows
...
Often the otter rolls over
in the water to clear its chest of any waste scraps and avoid soiling its coat
...
We hope you like this little story about these cute animals
...
8
Introduction
Product Overview
...
14
Methods described in this manual
...
18
High Pure Kits and
Silica Adsorption
High Pure PCR Template Preparation Kit
...
34
High Pure PCR Product Purification Kit
...
49
High Pure RNA Isolation Kit
...
64
High Pure FFPE RNA Micro Kit
...
75
High Pure miRNA Isolation Kit
...
95
High Pure Viral Nucleic Acid Kit
...
105
...
113
Agarose Gel DNA Extraction Kit
...
127
3
Chapter 3
Overview of Ion Exchange Chromatography
...
136
Genopure Plasmid Maxi Kit
...
150
Chapter 4
Overview of Solution-based Isolation
...
154
DNA Isolation Kit for Cells and Tissues
...
170
Chapter 5
Overview of Affinity Purification
...
184
mRNA Isolation Kit
...
197
mRNA Isolation Kit for Blood/Bone Marrow
...
208
Gel Filtration
Quick Spin Columns
...
214
Chapter 7
Automated Nucleic Acid
Isolation
4
MagNA Lyser Instrument
...
222
...
223
Chapter 8
Introduction
...
227
RT-PCR Product Selection Guide
...
229
Master Mixes
...
231
Reagents for Real-Time PCR
...
234
Appendix
Conversion Tables and Formulas
...
240
Other Useful Information
...
246
Ordering Guide
Isolation and Purification of RNA
...
248
Automated Isolation using the MagNA Pure LC Instrument
...
Automated Isolation using the MagNA Pure Compact
Instrument
...
Companion Reagents for Isolating Nucleic Acids
...
255
Index
5
Disclaimer
* For life science research only
...
+ For general laboratory use
...
Nucleic Subtype
Acid
Type
Origin/Source
Scale
Recommended Product
tissue, cultured cells, bacteria, yeast, blood,
dried blood spots
High Pure PCR Template Preparation Kit
DNA Isolation Kit for Cells and Tissues
propagated in E
...
coli
serum, plasma, blood, other body fluids,
s
upernatant from cell cultures
serum, plasma, whole blood
DNA
propagated in E
...
lysis
200 min, incl
...
lyticase digest
35 min, incl
...
lysis
High Pure PCR Product Purification Kit
PCR, modifying, labeling, restriction
digestion reaction, 100 µl; agarose gel
slices
>80% recovery of 5 – 25 µg DNA
>100 bp
10 min
High Pure PCR Cleanup Micro Kit
PCR, modifying, labeling, restriction
digestion reaction, agarose gel slices
>80% recovery of 5 – 25 µg DNA
10 min
High Pure Plasmid Isolation Kit*
E
...
coli HB 101, pUC 19 (2 ml)
E
...
5 µg
30 min
30 min
30 min
High Pure RNA Isolation Kit*
blood, 200 – 500 µl
cultured cells, 106
yeast, 108 cells
bacteria, 109 cells
sufficient for 10 RT-PCR reactions
20 µg
20 µg
35 – 50 µg
50 min, incl
...
lyticase digest
90 min, incl
...
5 – 3
...
5 ml
25 min
High Pure RNA Paraffin Kit
fresh-frozen or formalin-fixed paraffinembedded tissue
0
...
5 µg/5 µm section,
2‑6 µg/20 mg
fresh-frozen tissue
2 h without overnight
i
ncubation
High Pure FFPE RNA Micro Kit
1-10 µm FFPE tissue sections
1
...
5 µg/5 µm
60 min without 3h
incubation
Agarose Gel DNA Extraction Kit
agarose gel slices, 100-200 mg
80% recovery of fragments
(0
...
5 kb)
60 min
Genopure Plasmid Midi Kit
E
...
coli DH5, pBS (150 ml)
>420 µg
75 min
mRNA Isolation Kit for Blood/
Bone Marrow*
blood, bone marrow aspirate,
1
...
5 mg
7 – 14 µg/100 mg tissue
0
...
5 h, excl
...
5 h, excl
...
5 h, excl
...
5 h
2
...
5 h
3
...
As a plethora of methods exists for extraction and purification of nucleic acid, researchers usually choose the technique most suited to their:
Target nucleic acid (ssDNA, dsDNA, total RNA, mRNA, etc
...
)
Starting material (whole organ, tissue, cell culture, blood, etc
...
)
ownstream application (PCR, cloning, labeling, blotting, RT-PCR, cDNA synthesis,
D
RNase protection assays, etc
...
Often, the ideal lysis procedure is a compromise
...
g
...
Common lysis procedures include:
echanical disruption (for example, grinding, hypotonic lysis)
M
hemical treatment (for example, detergent lysis, chaotropic agents, thiol reduction)
C
nzymatic digestion (for example, proteases)
E
Cell membrane disruption and inactivation of intracellular nucleases may be combined
...
After cell lysis and nuclease inactivation, cellular debris may easily be removed by
filtration or precipitation
...
Extraction/precipitation
Solvent extraction is often used to eliminate contaminants from nucleic acids
...
Selective precipitation can also purify nucleic acids
...
Precipitation may also be used to concentrate nucleic acids
...
If the amount of target
nucleic acid is low, an inert carrier (such as glycogen) can be added to the mixture to
increase precipitation efficiency
...
Gel filtration exploits the molecular sieving properties of porous gel particles
...
Thus,
molecules are eluted in order of decreasing molecular size
...
The technique allows concentration and separation of molecules from a large volume in a short time
...
1
In adsorption chromatography, nucleic acids adsorb selectively onto silica or glass in the
presence of chaotropic salts, while other biological molecules do not
...
Affinity chromatography is a highly specific adaptation of adsorption chromatography
...
Washes then remove unbound components (with different structures)
...
Centrifugation
Selective centrifugation is a powerful purification method
...
Frequently, centrifugation is combined with another method
...
), for buffer exchange, or for size selection
...
Electrophoresis
Nucleic acids may be separated electrophoretically according to their size
...
In the presence of ethidium bromide, the
separated nucleic acids may be seen under UV light
...
For example, after PCR, electrophoresis is used to quickly check product length and
purity (absence of byproducts)
...
For instance, poly(A)+ mRNA may be
bound to streptavidin-coated magnetic particles by biotin-labeled oligo(dT) and the
particle complex removed from the solution (and unbound contaminants) with a
magnet
...
Introduction
15
Methods described in this manual
Methods described in this manual
1
This manual describes all the products Roche Applied Science currently sells for manual
nucleic acid purification and isolation
...
These rapid purification kits
eliminate traditional solvent extraction, precipitation, and electrophoresis steps
...
Solutions are just poured or pipetted into the matrix-filled columns
which are run by gravity flow
...
olution-based isolation uses proprietary cell lysis and extraction methods that are
S
quicker and safer than standard methods
...
ffinity purification exploits the hybridization properties of nucleic acids
...
el filtration relies on “spin columns” that are ready-to-use
...
All these products combine proven, reliable nucleic acid purification methods with
Roche Applied Science talent for optimization and innovation
...
For detailed
information please refer to www
...
com
...
All of them use methods that:
Can process multiple samples in minutes, rather than hours or days
2
Require less handling of potentially hazardous samples
Eliminate phenol extraction, precipitation, and other nucleic acid handling steps that
can lead to loss or fragmentation of the desired product
For a quick overview of these products, continue reading this article
...
5 – 4
...
coli
49
High Pure RNA Isolation Kit Intact total RNA from small amounts of whole blood,
cultured cells, yeast, gram positive or gram negative
bacteria
High Pure RNA Tissue Kit
Intact total RNA from tissues
64
High Pure FFPE RNA Micro
Kit
Total RNA from 1 – 10 µm FFPE tissue sections
69
High Pure RNA Paraffin Kit
Total RNA from fresh-frozen and formalin-fixed,
paraffin-embedded tissue sections up to 20 µm
75
High Pure miRNA Isolation
Kit
Tissue, stabilized tissue, cell culture, FFPE tissue
sections
84
High Pure Viral RNA Kit
Intact viral RNA from 200 – 600 µl of serum, plasma,
cell culture supernatant, tears, urine, or breast milk
95
High Pure Viral Nucleic
Acid Kit
Total viral nucleic acids (DNA and RNA) from
200 – 600 µl of serum, plasma, whole blood, or cell
culture supernatant
100
High Pure Viral Nucleic
Acid Large Volume Kit
Total viral nucleic acids (DNA and RNA) from
1 - 2
...
4 – 100 kb) from 100 – 200 mg
agarose gel slice
122
High Pure 96 UF Cleanup
Kit
18
55
Product DNA by high throughput ultrafiltration
127
Nucleic Acid Isolation and Purification Manual
Overview of Silica Adsorption
Principle of silica adsorption
All the kits described in this chapter depend on the tendency of nucleic acids to adsorb
to silica (glass) in the presence of a chaotropic salt such as sodium iodide (NaI), guanidine thiocyanate or guanidine hydrochloride (Melzak et al
...
This tendency was discovered by Vogelstein and Gillespie (1979) who found that DNA
fragments adsorbed to powdered flint glass in the presence of saturated NaI
...
, 1982)
Single-stranded phage nucleic acids (Kristensen et al
...
,
1989)
Genomic DNA (Yamada et al
...
, 1993)
Total RNA (Yamada et al
...
, 1990)
Different types of nucleic acid adsorb more or less tightly to glass depending on the ionic
strength and the pH of the surrounding solution
...
In each kit, this method is optimized to
prepare a particular type of nucleic acid
...
The glass fleece filter:
Adsorbs only nucleic acid, ensuring separation of the target molecules from a
complex biological mixture of proteins, sugars, lipids, and other components
Can be inserted into a microcentrifuge tube and processed in a standard tabletop
microcentrifuge
Allows processing of 0
...
5 ml samples in a series of centrifugation steps
specially constructed to ensure that contaminants suspended in the Wash Buffer
Is
are not retained by the filter or transferred to the eluted, purified nucleic acid
Benefit from smart column design
The High Pure Micro Column
Achieve high purity
...
Up to 10 µg binding capacity for use in demanding downstream
applications
...
The High Pure Mini Column
Perform long template applications
...
Maximize performance and accuracy in downstream assays
...
Obtain accurate results
...
High Pure Kits and Silica Adsorption
19
Overview of Silica Adsorption
The High Pure Extender Assembly
2
Benefit from smart column design using a removable High Pure mini spin
column
...
Use sample volumes up to 2
...
Obtain high-purity nucleic acids
...
Increase convenience and improve time to result
...
In each High Pure kit, the steps are basically the same and require only a few minutes
...
Substance added to serum
Highest concentration tested with no inhibition in
PCR after High Pure purification
Citrate
30 mg/ml
EDTA
300 mg/ml
Heparin
30 U/ml
Hemoglobin
50 mg/ml
Performance of High Pure Nucleic Acid Purification in the removal of different anticoagulants or human
hemoglobin
...
Instead of glass fiber fleece (as in the High Pure kits), one can also use silica beads to
adsorb DNA
...
In each, the nucleic acid is adsorbed to silica in the presence of a
chaotropic salt, pelleted by centrifugation (while adsorbed to the silica beads), washed
extensively to remove contaminants, then released from the beads with a low salt buffer
...
References
Boom, R
...
J
...
, Salimans, M
...
M
...
L
...
M
...
and
van der Noordaa, J
...
Clin
...
28, 495 – 503
Kristensen, T
...
and Ansorge, W
...
15, 5507 – 5516
Marko, M
...
, Chipperfield, R
...
C
...
Biochem
...
A
...
S
...
F
...
and Haynes, C
...
(1996) J
...
(USA) 181, 635 – 644
Vogelstein, B
...
(1979) Proc
...
Acad
...
USA 76, 615 – 619
Yamada, O
...
, Nakashima, M
...
, Kamahora, T
...
, Kishi,
Y
...
and Kurimura, T
...
Virol
...
27, 203 – 210
Zeillinger, R
...
, Speiser, P
...
(1993) Biotechniques 14,
202 – 203
Zimmermann, J
...
, Kristensen, T
...
, Stegemann, J
...
and
Ansorge, W
...
Cell
...
1, 29 – 34
20
Nucleic Acid Isolation and Purification Manual
High Pure PCR Template Preparation Kit
High Pure PCR Template Preparation
Kit
for preparation of up to 100 nucleic acid samples
Cat
...
11 796 828 001
Principle
Cells are lysed during a short incubation with Proteinase K in the presence of a chaotropic salt (guanidine HCl), which immediately inactivates all nucleases
...
The nucleic acids
remain bound while a series of rapid “wash-and-spin” steps remove contaminating small
molecules
...
The process does not require precipitation, organic solvent extractions, or extensive
handling of the nucleic acids
...
2 – 0
...
Time required
Total time: approx
...
12 min (for whole blood or cultured cells)
Results
Yield: Variable, depending on sample type (See the table under Part IV of “How to
use the kit” in this article)
...
RNA can be removed from the purified nucleic acids with an optional RNase
digestion (see page 24)
...
Improves PCR reproducibility and reliability, because the kit removes inhibitors that
might cause PCR templates to behave unpredictably
...
Ideal for a wide variety of research projects, because one kit can purify nucleic acids
from many sources
...
Flow diagram
The chart shows as an example protocol steps for the sample material whole blood
...
Kit contents
Tissue Lysis Buffer with 4 M urea (20 ml)
Binding Buffer with 6 M guanidine HCl (20 ml)
Proteinase K, lyophilized recombinant
Dissolve in 4
...
Store aliquots at –15 to –25°C
...
Wash Buffer (20 ml)
Add 80 ml absolute ethanol to Wash Buffer before use
...
5; 40 ml)
Warm the Elution Buffer to +70°C before use
...
Additional materials needed
2
Absolute ethanol
Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force
Microcentrifuge tubes, 1
...
Typical nucleic acid yield from different organisms
(research samples)
Starting material
Sample size
Yield (µg)
Whole blood, human
200 µl*
3 – 6 (DNA)
Buffy coat
200 µl*
20 (DNA
Cultured cells (K562)
10 cells
15 – 20 (DNA)
Calf thymus
25 mg
5 – 10 (DNA)
Mouse tail
0
...
5 cm (25 – 50 mg)
5 – 10 (DNA)
Yeast cells
10 cells
10 – 13 (DNA)
Bacteria cells
10 cells
1 – 3 (total nucleic acids)
Dried blood spots
9 mm punch-out
Detectable in PCR
6
8
9
* ypical volume is 200 µl; maximum volume is 300 µl
...
V
...
solation of nucleic acids from 200 µl whole blood, 200 µl buffy
I
coat, or 104 – 105 cultured mammalian cells
If the sample is <200 µl, add PBS to make the total volume 200 µl
...
Maximum sample
volume is 300 µl
...
5 ml microcentrifuge tube:
E Add 200 µl sample
...
E Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
EEE
High Pure Kits and Silica Adsorption
23
High Pure PCR Template Preparation Kit
How to use the kit
After the incubation, mix sample with 100 µl isopropanol
...
E Pipette entire sample into upper buffer reservoir of the Filter Tube
...
After centrifugation:
E Remove the Filter Tube from the Collection Tube and discard the liquid and the
Collection Tube
...
Repeat the centrifugation step (1 min at 8000 x g) and discard
flowthrough and Collection Tube
...
To wash the sample:
E Add 500 µl Wash Buffer to the upper reservoir of the Filter Tube
...
After the first wash:
E Repeat Step 5
...
Discard flowthrough
...
13,000 x g) to remove residual Wash Buffer
...
5 ml
microcentrifuge tube
...
E Centrifuge the tube assembly for 1 min at 8000 x g
...
The microcentrifuge tube now contains the eluted nucleic acids
...
E OR store the eluted nucleic acids at +2 to +8°C for later analysis
...
No
...
For example, add 0
...
For nucleic
acids from 107 cells, add 1
...
For nucleic acids
from 108 cells, add 16 µl RNase and incubate 30 min at +37°C
...
24
Nucleic Acid Isolation and Purification Manual
High Pure PCR Template Preparation Kit
How to use the kit
Vb
...
We recommend the use a 9 mm punch out of
a Whatman 903 Specimen Collection Paper
1
To a sterile 1
...
E Add 200 µl Tissue Lysis Buffer
2
Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
Add 200 µl Binding Buffer
...
To transfer the sample to a High Pure Tube:
Insert one High Pure Filter Tube into one Collection Tube
...
Take care not to block the pipette tip by the card punches
...
After centrifugation:
Remove the Filter Tube from the Collection Tube and discard the liquid and the
Collection Tube
...
Repeat the centrifugation step (1 min at 8000 x g) and discard
flowthrough and Collection Tube
...
To wash the sample:
Add 500 µl Wash Buffer to the upper reservoir of the Filter Tube
...
After the first wash:
Repeat Step 5
...
Discard flowthrough
...
13,000 x g) to remove residual Wash Buffer
...
5 ml
microcentrifuge tube
...
Centrifuge the tube assembly for 1 min at 8000 x g
...
The microcentrifuge tube now contains the eluted nucleic acids
...
OR store the eluted nucleic acids at +2 to +8°C for later analysis
...
Isolation of nucleic acids from 25 – 50 mg mammalian tissue
To a clean, sterile 1
...
To increase the yield of nucleic acids, cut the sample into small pieces with a
scalpel
...
2
E Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
After tissue lysis:
E Add 200 µl Binding Buffer to the tube and mix well
...
Add 100 µl isopropanol to the tube, then:
E Mix the contents of the tube
...
This step draws insoluble tissue segments into the pipette tip and blocks it
...
E Pipette the remainder of the liquid sample into the upper reservoir of a combined
Filter Tube-Collection Tube assembly
...
K
Vd
...
5 ml microcentrifuge tube:
E Add pieces from 0
...
5 cm (25 – 50 mg) mouse tail
...
E Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
Use a 1 ml disposable syringe without needle to shear the lysed tail sample:
E Draw the sample into the syringe and then expel it again
...
To the sheared sample:
E Add 200 µl Binding Buffer
...
E Centrifuge the tube for 5 min at 13,000 x g
...
E Follow Protocol Va page 24, starting at the first centrifugation step (Step 4)
...
Isolation of nucleic acids from 109 bacteria or 108 yeast cells
In a clean, sterile 1
...
E Resuspend the cell pellet in 200 µl PBS
...
E If the sample is yeast, go to Step 3
...
0)
...
E Go to Step 4
...
5 mg/ml)
...
E Go to Step 4
...
E Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
Follow Protocol Va page 24, starting at the addition of isopropanol (Step 2)
...
Data (see page 31) of cell lysis conditions for lactobacilli
and for staphylococci are kindly provided by C
...
A
...
Hertel, University of Hohenheim, Germany
...
Isolation of nucleic acids from a formalin-fixed paraffin-embedded
tissue section
[Protocol was kindly provided by T
...
30 min, to deparaffinize it
...
Incubate the tissue section for 10 s in each of the following:
E Absolute ethanol (dehydration)
The section should turn white after it is transferred to ethanol
...
E Transfer the sample to a clean, sterile, preweighed 1
...
E Determine the weight of the sample
...
E Add 40 µl reconstituted Proteinase K solution and mix the contents of the tube
immediately
...
Optimal incubation time will depend on the type of tissue
...
2
After the first incubation:
E Again, add 20 µl reconstituted Proteinase K solution
...
After this incubation step, no crude tissue particles should be visible
...
E Incubate for 10 min at +70°C
...
E Use an automatic pipette to draw part of the sample into a 1 ml pipette tip
...
E Withdraw the pipette tip, carrying the insoluble tissue segments with it
...
Follow Protocol Va, page 24, starting at the first centrifugation step (Step 4)
...
Isolation of mycobacterial DNA
(Protocol was kindly provided by B
...
Reischl, University of Regensburg, Germany)
100 µl decontaminated research sample material (respiratory samples, nonrespiratory
samples or sediments from liquid Kirchner media) is centrifuged for 10 min at
14,000 x g
...
Add 200 µl PBS to the pellet and resuspend by vortexing
...
Incubate for 10 min at +70°C
...
In total the samples are boiled 6 times and frozen 5 times
...
Add 100 µl isopropanol and mix by vortexing
...
VI
...
For details on
how to troubleshoot the above protocols, see the General Troubleshooting Procedure for
all High Pure kits on page 113 of this manual
...
28
Nucleic Acid Isolation and Purification Manual
High Pure PCR Template Preparation Kit
Typical results with the kit
Typical results with the kit
Experiment 1
6
...
3 µg 1 µg
µg 0
...
Nucleic acids were prepared from blood or cultured
human K562 cells according to Protocol Va, and 250 ng of each
preparation were amplified by Expand Long Template PCR
...
The PCR cycling conditions
and the sequence of the tPA primers were as described in the
Expand Long Template PCR package insert
...
3 kb: Obtained from blood, amplified in Expand Long
Template PCR buffer 1
Lane 3: 15 kb: Obtained from blood, amplified in Expand Long
Template PCR buffer 3
Lane 4: 23 kb: Obtained from blood, amplified in Expand Long
Template PCR buffer 3
Lane 5: 28 kb: Obtained from K562 cells, amplified with the
Expand 20 kbPLUS System
Result: All preparations yielded a distinct specific band of the
expected fragment length even as large as 28 kb from genomic
DNA prepared with the High Pure PCR Template Preparation Kit
...
Nucleic acids were purified from cultured
mammalian cells (106 K562 cells) by Protocol Va and from calf
thymus tissue (25 mg) by Protocol Vb
...
The blot was hybridized to a DIG-labeled b-actin
antisense RNA probe according to standard procedures
...
The blot was exposed to X-ray film for 1 h
...
3 µg, 1 µg, and 3 µg samples of K562 genomic
DNA, purified by traditional methods (phenol/chloroform
extraction, ethanol precipitation) after Eco RI digestion
Lanes 7, 8: 0
...
High Pure Kits and Silica Adsorption
29
High Pure PCR Template Preparation Kit
Typical results with the kit
Experiment 3
F
igure 3: Use of nucleic acids from
paraffin-embedded tissue (prepared with
the High Pure PCR Template Preparation Kit)
for amplification of the human androgen
receptor gene
...
An aliquot
(10 µl) of each nucleic acid product was used as
template for the Expand High Fidelity PCR
System (100 µl reaction mixture)
...
Aliquots (20 µl) of each amplicon
were separated electrophoretically and the gel
was stained with ethidium bromide
...
Experiment 4
Incubation period
15 min
30 min
60 min
L
...
25 U/µl Mu
0
...
2 (40
...
3 (40
...
4 (43
...
2 (53
...
6 (64
...
0 (100 %)
21
...
4 %)
24
...
2 %)
30
...
0 %)
L
...
25 U/µl Mu
0
...
2 (10
...
9 (29
...
0 (53
...
7 (17
...
3 (70
...
5 (100 %)
26
...
2 %)
70
...
7 %)
131
...
6 %)
L
...
25 U/µl Mu
0
...
5 (11
...
0 (29
...
3 (67
...
6 (14
...
8 (60
...
4 (100 %)
52
...
7 %)
168
...
1 %)
196
...
6 %)
S
...
3 (10
...
5 (17
...
2 (17
...
5 (31
...
0 (99
...
5 (100 %)
64
...
2 %)
105
...
2 %)
106
...
7 %)
Table 1: Use special lysis conditions for gram-positive bacteria to achieve high DNA concentrations
(µg/ml) in eluate
...
The
percentages refer to the DNA yield obtained with the highest mutanolysin or lysostaphin concentration and an
incubation period of 30 min
...
aureus (A) and for various lactobacilli (B)
A:
Lanes 1 and 12: Molecular Weight Marker 1 kb ladder
Lane 2: Negative control (without DNA)
Lanes 3 – 5: S
...
aureus; lysis with 10 µg/ml lysostaphin,
i
ncubation periods 15, 30 and 60 min
Lanes 9 – 11: S
...
casei
Lane 15: L
...
sakei
Lane 17: Negative control (without DNA)
Experiment 5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 LS
VIII
RPCJE
F
igure 5: Gel electrophoresis of PCR products and controls
...
tuberculosis infection
Lane 2: Smear –; Specimen sputum
Lane 3: Smear –; Specimen feces
Lane 4: Smear –; Specimen feces; double because of
different wash step
Lane 5: Smear +/–; Specimen sputum
Lane 6: Smear +; Specimen BAL
Lane 7: Smear ++; Specimen sputum; COBAS®
AMPLICOR® MTB +
Lane 8: Smear ++; Specimen sputum; known
M
...
avium infection
Lane 10: Smear +; Specimen sputum
Lane 11: Smear +; Specimen liquid Kirchner media
sediment; Smear: acid-fast, rod-shaped
Lanes 12 – 22: Internal controls of samples of
lane 1 – 11
Lane 23: Negative control
Lane 24: Positive control M
...
The eluted DNA was analyzed by amplification detection in M
...
To control the successful amplification an agarose gel electrophoresis was
performed
...
tuberculosis H37 DNA) are positive (lanes 12 – 22, 242 bp amplification
product)
...
Samples of lanes 1, 5, 6, 7, 10
are assessed as positive which is in accordance with the corresponding smear results
...
Amplification has been performed in LightCycler® capillaries using SYBR Green and
Hybridization Probes as the detection formats
...
2
Sample
Volume/Amount
Range
Typical
concentration
range [ng/µl]
Eluate use in
LightCycler®
PCR (µl)
Whole blood human*
1 – 50 µl **
0
...
5 ng – 125 ng]
Cultured cells, K562*
100 – 105 cells
0
...
01 – 50 ng]
* Research samples
** Yield may vary between different blood donors because they may have different amounts of leukocytes
Procedure modification
The standard procedure for whole blood and cultured cells has been used for
LightCycler® sample preparation except the elution volume was set to 50 µl in order to
increase nucleic acid concentration for minute sample amounts
...
Parameter
Sample
Material
SYBR Green
Min
cyclophilin A
blood
0
...
005 µl
100 µl
10
1
105
4
Max
a)
a)
The above table shows the minimal and maximal sample amounts which have been
purified and used for LightCycler® Amplification with whole blood and cultured cells as
sample material
...
All values are for 20 µl LightCycler® Amplification when 5 µl of 50 µl total eluate
is applied
...
32
Nucleic Acid Isolation and Purification Manual
High Pure PCR Template Preparation Kit
References
Barré, L
...
(2007) Am J Physiol Endocrinol Metab, 292, E802 – E811
Budak, F
...
(2007) Int J Neurosci, 117(3), 409 – 15
Bulst, S
...
(2009) Hum
...
Genet
...
et al
...
Clin
...
45, 1673 – 1678
Daly, K
...
et al
...
Bacteriol
...
et al
...
, 70, 7304 - 7314
Fassina, A
...
(2009) J
...
Pathol
...
et al
...
Biol
...
, 24, 269 – 280
Gomes, J
...
et al (2007) Genome Res
...
A
...
(2009) Infect
...
, 77, 3894 – 3901
2
Lau, A
...
(2010) J
...
Microbiol
...
et al
...
Clin
...
, 45, 906 – 914
Talaulikar, D
...
(2008) J
...
Pathol
...
No
...
No
...
Since the binding process is specific for nucleic acid, the bound material can be
separated and purified from impurities by a simple wash step
...
Small oligonucleotides and
dimerized primers from amplification reactions are selectively removed
...
Starting material
Samples (up to 100 µl each) could contain:
Amplified DNA products that are between 50 bp and 5 kb long
Modified DNA fragments [e
...
, DNA processed with restriction enzymes, T4 polymerase or other enzymes] that are between 50 bp and 5 kb long
Hapten-labeled (e
...
, DIG-labeled) or fluorescently labeled DNA fragments
RNA from in vitro transcription reactions
First and second strand cDNA
Samples (up to 100 mg) of agarose gel slices
Application
Use the High Pure PCR Cleanup Micro Kit to efficiently purify products from PCR and
other reactions
...
g
...
In addition, the kit can be utilized to purify cDNA,
concentrate dilute nucleic acid solutions, and recover DNA from agarose gel slices
...
For 100 µl liquid sample or
100 mg agarose gel slice
Purification of
liquid sample,
100 µl
Purification from
Agarose Gel,
100 mg
Removal of low
molecular DNA
34
Labeling or other
reaction products
100 bp to 5 kb
PCR products
50 bp to 5 kb
DNA fragments
for sequencing
DNA fragments
100 bp to 5 kb
Primer up to 25
bases
Primer-Dimer up
to 70 bp
300 µl Binding Buffer +
200 µl Binding Buffer +
200 µl Binding Enhancer 100 µl Binding Enhancer
(20%)
(40%)
+
400 µl Binding
Buffer
+
+
+
+
+
Nucleic Acid Isolation and Purification Manual
High Pure PCR Cleanup Micro Kit
Time required
The entire High Pure PCR Cleanup Micro Kit method takes approx
...
Results
The amount of DNA recovered is dependent on the amount of DNA applied to the glass
fiber fleece, the elution volume, and the length of the amplification/DNA products
...
Benefits
Conserve resources by using one versatile kit that eliminates the need to use several
kits from other suppliers
...
Obtain purified product in a small elution volume (10 μl) for demanding downstream applications
...
Generate contaminant-free DNA for direct use in cloning, ligation, restriction
digests, and other reactions
...
High Pure Kits and Silica Adsorption
35
High Pure PCR Cleanup Micro Kit
How to use the kit
How to use the kit
I
...
speed
for 1 min
Discard collection tube
Centrifuge at 8,000 x g
for 1 min
Connect new tube;
add 10 – 20 µl Elution Buffer
to the middle of the filter
Purified PCR or other
reaction product
* Please select from the Application Selection Guide of the pack insert
...
Kit contents
Binding Buffer 20 ml or 80 ml
Binding Enhancer 15 ml or 45 ml
Wash Buffer 10 ml or 2 x 20 ml
Elution Buffer 40 ml
High Pure Micro Filter Tubes (50 or 200 tubes)
Collection Tubes (50 or 200 tubes)
36
Nucleic Acid Isolation and Purification Manual
High Pure PCR Cleanup Micro Kit
How to use the kit
III
...
5 ml, sterile
Do not use vessels or pipettes containing polystyrene (PS) when working with the
Binding Enhancer (bottle 2)
...
4 or
TBE buffer (89 mM Tris-borate, 2 mM EDTA) pH 8
...
5 ml, sterile
IVa
...
For other applications please refer to Application Selection
Guide in the pack insert
To process a larger sample (>100 µl), divide it into several 100 µl aliquots and
process each as a separate sample
...
If the
sample is > 100 µl, it should be in a 1
...
1
After the PCR is finished, adjust total volume of each sample to 100 µl by adding
PCR grade water
...
g
...
E Centrifuge the mixture briefly
...
E Using a pipette, transfer the sample from step 1 to the upper reservoir of the Filter
Tube
...
3
Disconnect the Filter Tube, and discard the flowthrough solution
...
4
Add 400 µl Wash Buffer to the upper reservoir
...
EEE
High Pure Kits and Silica Adsorption
37
High Pure PCR Cleanup Micro Kit
How to use the kit
5
Discard the flowthrough solution
...
E Add 300 µl Wash Buffer
...
This second (300 µl) wash step ensures optimal purity
...
E Reconnect the Filter Tube to the same Collection Tube
...
E Discard the flowthrough solution and the Collection Tube
...
5 ml microcentrifuge tube
...
7
Add 10 - 20 µl Elution Buffer to the center of the Filter Tube
...
Do not use water for elution, since alkaline pH is required for optimal yield
...
If you plan to determine the A260 of the eluted DNA, first centrifuge the eluate
for more than 1 min at maximum speed to remove residual glass fibers, which
may interfere with the absorbance measurement
...
K
IVb
...
1
Isolate DNA band of interest via agarose gel electrophoresis as follows:
E Load PCR product mixture on a 0
...
E Use 1 x TAE or 1 x TBE as running buffer
...
2
Identify bands by staining gel with ethidium bromide or SYBR Green I Nucleic Acid
Gel Stain
...
3
Cut desired DNA band from gel using a scalpel or razor blade that has been sterilized
with ethanol
...
4
Preweigh an empty, sterile 1
...
E Place excised agarose gel slice in the sterile microcentrifuge tube
...
5
To the microcentrifuge tube, add 300 µl Binding Buffer for every 100 mg agarose gel
in the tube
...
E Vortex the microcentrifuge tube 15 - 30 s to resuspend the gel slice in the Binding
Buffer
...
E Vortex the tube briefly every 2 - 3 min during incubation
...
E Vortex thoroughly
...
8
Insert one High Pure Filter Tube into one Collection Tube
...
E Centrifuge 30-60 s at 8000 x g in a standard table top centrifuge at +15 to +25°C
...
Troubleshooting the High Pure protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
For factors that may affect the High Pure
PCR Cleanup Micro Kit, see page 115
...
Lane 1: Molecular weight marker VI
Lane 2: 0% Binding Enhancer
Lane 3: 10% Binding Enhancer
Lane 4: 20% Binding Enhancer
Lane 5: 40% Binding Enhancer
Lane 6: PCR without purification
Lane 7: PCR negative control (PCR without template)
Lane 8: Molecular weight marker VI
Experiment 2
1
2
3
4
5
6
7
— 110
— 100
— 90
— 80
— 70
— 60
— 50
— 40
Figure 7: Electropherogram of the DNA fragment distribution obtained from an Agilent Bioanalyzer run (Agilent,
USA)
...
et al
...
, 70: 9423 - 9433
Eno C
...
et al
...
Bull
...
et al
...
et al
...
Agents Chemother
...
et al
...
H
...
(2009) J
...
Chemother
...
et al
...
Clin
...
, 48: 2902 - 2908
Steiner R
...
et al
...
Natl
...
Sci
...
(2010) J
...
Chem
...
No
...
No
...
The DNA remains bound while a series of rapid “wash-and-spin”
steps remove contaminating small molecules (including small nucleic acids)
...
The process does not require
DNA precipitation, organic solvent extractions, or extensive handling
...
Time required
Total time: approx
...
Purity: Purified DNA is free of short DNA (<100 bp), small molecules (for example,
mineral oil, primers, salts, unincorporated nucleotides) and proteins (for example,
thermostable enzymes)
...
Minimizes DNA loss, because the kit removes contaminants without precipitation or
other handling steps that lead to DNA loss or degradation
...
Accommodates a wide variety of samples, because the kit can purify DNA from
most enzymatic reaction mixtures, DNA from agarose gels, even RNA from in vitro
transcription reactions
...
42
Nucleic Acid Isolation and Purification Manual
High Pure PCR Product Purification Kit
How to use the kit
How to use the kit
I
...
speed
for 30 – 60 s
Add 500 µl Wash Buffer
Discard flowthrough
Centrifuge at 13,000 x g
for 1 min
2
Add 200 µl Wash Buffer
Discard flowthrough
Centrifuge at 13,000 x g
for 1 min
Discard flowthrough
and Collection Tube
Add 50 – 100 µl
Elution Buffer
Centrifuge at 13,000 x g
for 1 min
Purified PCR Product
II
...
5) (40 ml)
High Pure Filter Tubes (50 or 250 tubes)
Collection Tubes, 2 ml (50 or 250 tubes)
Both sizes of the kit contain the same components; only the amount of the components in the kit changes
...
Additional materials needed
Absolute ethanol
Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force
Microcentrifuge tubes, 1
...
0
TBE Buffer (89 mM Tris-borate, 2 mM EDTA) pH 7
...
Expected DNA recovery from various amounts and sizes
of DNA
DNA applied
(µg)
Recovery (%)
Fragment1,2
length (bp)
Recovery (%)
Elution
volume1 (µl)
Recovery (%)
5
2
77
<100
< 5
50
68
10
79
375
>95
100
79
25
80
700
>95
150
80
50
56
3000
>95
200
80
1
2
10 µg DNA was used for this experiment
...
V
...
Purifying amplification products from 100 µl PCR product mix
To process a larger sample (>100 µl), either increase proportionally the amount of
Binding Buffer (Step 1) or divide the large sample into several aliquots and process
the aliquots as separate samples
...
You do not need to remove mineral oil or wax from the PCR solution before
adding the Binding Buffer
...
2
After mixing the sample:
E Insert one High Pure Filter Tube into one Collection Tube
...
3
Insert the entire High Pure Tube assembly into a standard tabletop microcentrifuge,
then centrifuge the tube assembly for 30 s at full speed (approx
...
4
After the centrifugation:
E Remove the Filter Tube from the Collection Tube
...
E Reinsert the Filter Tube in the same Collection Tube
...
E Repeat the centrifugation (as in Step 3)
...
E Add 200 µl Wash Buffer to the upper reservoir of the Filter Tube
...
EEE
44
Nucleic Acid Isolation and Purification Manual
High Pure PCR Product Purification Kit
How to use the kit
7
After the second wash:
E Remove the Filter Tube from the Collection Tube
...
E Insert the Filter Tube into a clean, sterile 1
...
8
To elute the DNA from the Filter Tube:
E Add 50 – 100 µl Elution Buffer or PCR grade water (pH 8
...
5) to the upper
reservoir of the Filter Tube
...
E Centrifuge the tube assembly for 30 s at full speed
...
You may:
E EITHER use the eluted DNA directly in such applications as cloning or sequencing
E OR store the eluted DNA at +2 to +8°C for later analysis
...
Purifying DNA from a 100 mg slice of agarose gel
1
Purify the DNA of interest electrophoretically as follows:
E Prepare an agarose gel with Roche Applied Science agarose (MP, LE or MS) and
load the DNA of interest on the gel
...
0) or 1 x TBE (45 mM Trisborate, 1 mM EDTA, pH 8
...
E Electrophorese until the DNA of interest is well separated from contaminants
...
3
Cut the DNA band from the gel with a sharp scalpel or razor blade
...
4
Place the agarose gel slice into a sterile 1
...
5
To the microcentrifuge tube, add 300 µl Binding Buffer for every 100 mg agarose gel
...
E Incubate the suspension for 10 min at +56°C
...
7
After the gel is completely dissolved:
E To the microcentrifuge tube, add 150 µl isopropanol for every 100 mg agarose gel
...
8
After mixing the components of the microcentrifuge tube:
E Insert one High Pure Filter Tube into one Collection Tube
...
If the volume of gel suspension is >700 µl, divide the suspension into two
portions and use separate Filter Tubes for each portion
...
K
High Pure Kits and Silica Adsorption
45
High Pure PCR Product Purification Kit
Typical results with the kit
Vc
...
2
Add 500 µl Binding Buffer and mix well
...
When using other sample volumes than 100 µl, adjust the volume of Binding
Buffer accordingly
...
The elution efficiency is increased with higher volume of Elution Buffer applied
...
Normally, almost quantitative recovery can be found, as can be
determined in a direct detection assay
...
Troubleshooting the High Pure protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
For factors that may affect the High Pure
PCR Product Purification Kit, see page 116
...
9 kb p53
4
...
3 kb tPA
9
...
Four different long PCR products were generated with the Expand Long Template PCR System, purified by either
of two methods, then analyzed electrophoretically
...
9 kb fragment from the p53 gene; 4
...
3 kb, and 9
...
For each size product, the gel shows:
Lane 1: DNA before purification
Lane 2: DNA after purification by phenol/chloroform extraction
Lane 3: DNA after purification with the High Pure PCR Product Purification Kit
Result: The High Pure Kit removed primers and primer dimers, while recovering 90% of the long PCR products
(as calculated by photospectroscopy)
...
Papenbrock, P
...
Schmidt, University of Hannover, Germany)
Background: Sulfurtransferases are a group of enzymes widely distributed in plants,
animals, and bacteria
...
In a database search several Arabidopsis EST clones with homologies to bacterial and
mammalian sulfurtransferases could be identified
...
The protein was expressed in
E
...
However, RNA expression data, protein contents obtained by Western blot
analysis and sulfurtransferase enzyme activity measurements using 3-mercaptopyruvate
or thiosulfate as substrates did not correlate with each other in Arabidopsis plants in
different developmental states
...
Different EST clones that show homology to sulfurtransferases and also to senescence-associated proteins are initially characterized
...
For preparing
the hybridization probes cDNAs of interest were separated from the vectors with the
respective restriction enzymes (MST1 coding for the mature 3-mercaptopyruvate sulfurtransferase, 950 bp, Kpn I/Pst I; EST 46D8T7, 600 bp, Sal I/ Not I)
...
The gel pieces containing the MST1 or the
46D8T7 fragment were eluted following exactly the inserted recipe for isolating DNA
from agarose gels using the High Pure PCR Product Purification Kit
...
Northern blot was performed as previously described
...
Results: Hybridization of restricted genomic Arabidopsis DNA with a digoxigenin-labeled
MST1 probe by random priming reveals the existence of at least two, and probably three
different sulfurtransferase genes in the Arabidopsis genome (Figure 9, right)
...
RNA homologous to the sequence of EST clone 46D8T7 was expressed in very young plants
...
In the plants of a later developmental stage, expression level parallels the
e
xpression of SAG 13 clone, the best marker of senescence known so far
...
Genomic DNA was restricted with BamH I, EcoR I, Hind III, and Xba I
overnight, transferred to nylon membrane and hybridized with a digoxigenin probe labeled by random prime
labeling
...
Right: cDNA from the EST clone 46D8T7 was labeled
...
Total RNA was extracted from Arabidopsis plants of different ages (from
the left to the right: harvested after 10, 14, 17, 21, 24, 28, 31, 35, 38, and 42 days), separated under denaturing
conditions and transferred to nylon membranes
...
Filters were hybridized with PCR fragments labeled with digoxigenin
...
PCR-probe (1300 bp) used for hybridizing the filter at the
bottom: cDNA from the SAG13 clone (senescence associated gene)
...
Purification of digoxigenin-labeled DNA via PCR using the High
Pure PCR Product Purification Kit seems to increase the sensitivity of probes and reduce
unspecific binding
...
(2010) Mol
...
Proteomics, 9: 2601 - 2616
Besseyre T
...
(2010) Infect
...
, 78, 260 – 264
Blas-Machado, U
...
(2007) Vet
...
, 44, 110 –115
Dotta, F
...
(2007) PNAS, 104, 5115 – 5120
Frias M
...
et al
...
Bacteriol
...
et al
...
Bacteriol
...
et al
...
Biol
...
, 282, 542 – 554
Martins E
...
et al
...
Bacteriol
...
H
...
(2009), Jpn
...
Clin
...
, 39, 509 – 513
Stucka, R
...
et al
...
J
...
Metab
...
No
...
No
...
Then, in the presence of a chaotropic salt (guanidine HCl), plasmid DNA binds
selectively to glass fiber fleece in a special centrifuge tube
...
Finally, low salt elution removes the DNA from the glass fiber fleece
...
Starting material
– 4
...
coli cultures research samples (at a density of 1
...
0
0
...
Time required
Total time: approx
...
coli strain (See the table under Part IV of “How
to use the kit” in this article)
...
Benefits
Saves time, because the kit can prepare up to 24 plasmid samples in less than
30 min
...
Increases lab safety, because the kit does not use hazardous organic reagents such as
cesium chloride, phenol, chloroform, or ethidium bromide
...
High Pure Kits and Silica Adsorption
49
High Pure Plasmid Isolation Kit
How to use the kit
How to use the kit
I
...
5 – 4 ml
E
...
Incubate at
+15 to +25°C
for 5 min
Add 350 µl chilled Binding
Buffer
Mix gently
...
Centrifuge at
max
...
speed
for 30 – 60 s
Add 500 µl Wash Buffer I
(optional)
Discard flowthrough
Centrifuge at 13,000 x g
for 1 min
Add 700 µl Wash Buffer II
Discard flowthrough
Centrifuge at 13,000 x g
for 1 min plus additional
min after discarding
flowthrough
Discard flowthrough
and Collection Tube
Add 100 µl Elution Buffer
Centrifuge at 13,000 x g
for 1 min
Purified Plasmid DNA
II
...
5 mg or 8 mg)
Dissolve RNase powder in Suspension Buffer before use
...
5) (40 ml)
High Pure Filter Tubes (50 or 250 tubes)
Collection Tubes, 2 ml (50 or 250 tubes)
Both sizes of the kit contain the same components; only the amount (values in
parentheses) of the components in the kit changes
...
Additional materials needed
Absolute ethanol
Centrifuge tubes and centrifuge for harvesting up to 4 ml bacterial culture
Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force
Microcentrifuge tubes, 1
...
Typical DNA recovery from various E
...
coli host strain/density
Culture volume
0
...
0 ml
2
...
0 ml
XL 1 blue (3
...
9 µg
8
...
8 µg
14
...
5 A600/ml)
0
...
7 µg
3
...
2 µg
HB 101 (4
...
8 µg
3
...
9 µg
8
...
Protocol for preparing DNA from 0
...
0 ml of E
...
Prepare the starting material:
E Pellet the bacterial cells from 0
...
0 ml of E
...
The cells should have a density of 1
...
0 A600 units per ml
...
E Add 250 µl Suspension Buffer + RNase to the centrifuge tube containing the
bacterial pellet
...
Treat the resuspended bacterial pellet as follows:
E Add 250 µl Lysis Buffer
...
To avoid shearing genomic DNA, do not vortex!
E Incubate for 5 min at +15 to +25°C
...
E Mix gently by inverting the tube 3 to 6 times
...
The solution should become cloudy and a flocculent precipitate should form
...
13,000 x g (full speed) in a standard tabletop
microcentrifuge
...
E Transfer entire supernatant from Step 5 into upper buffer reservoir of the Filter
Tube
...
E Centrifuge for 30 – 60 s at full speed
...
E Does the E
...
Example: XL 1 blue and DH 5 a do not have high nuclease activity and do not
require the optional wash with Wash Buffer I
...
Example: HB 101 and JM strains have high nuclease activity and require the
optional wash with Wash Buffer I
...
E Centrifuge for 30 – 60 s at full speed and discard the flowthrough
...
E Centrifuge for 30 – 60 s at full speed and discard the flowthrough
...
E Discard the Collection Tube
...
5 ml microcentrifuge tube
...
0 – 8
...
E Centrifuge the tube assembly for 30 – 60 s at full speed
...
You may:
E EITHER use the eluted DNA directly in such applications as cloning or sequencing
E OR store the eluted DNA at +2 to +8°C or –15 to –25°C for later analysis
...
Troubleshooting the High Pure protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
For factors that may affect the High Pure
Plasmid Isolation Kit, see page 116
...
A
derivative of pUC18 containing a 3
...
coli XL 1 blue (1
...
Isolated plasmid DNA (250 ng) was sequenced with fluorescent-labeled
sequencing primer under the following cycle sequencing conditions: initial denaturation, 2 min at +95°C; then,
repeated cycles of denaturation (30 s, +95°C), primer annealing (30 s, +60°C), and extension/termination (60 s,
+70°C)
...
3% PAA gel (66 cm plate)
...
Result: More than 700 nucleotides can be read with High Pure Plasmid Isolation Kit prepared sequencing
template on a LI-COR model 4000 S automated sequencing system using automated base calling and the
autostop function
...
et al
...
Med
...
, 58, 638 – 643
Binnicker, M
...
et al
...
Clin
...
, 45, 173 – 178
Cunningham S
...
et al
...
Clin
...
, 48: 2929 - 2933
Daly K
...
et al
...
Bacteriol
...
et al
...
Animicrob
...
, 59, 841 – 847
Grys T
...
(2009) J
...
Microbiol
...
K
...
et al
...
Antimicrob
...
, 61: 488 – 497
Manso I
...
(2009) J
...
Chem
...
et al
...
A
...
(2007) J
...
, 189, 4718 – 4728
Nucleic Acid Isolation and Purification Manual
High Pure RNA Isolation Kit
High Pure RNA Isolation Kit
for isolation of total RNA from up to 50 samples
Cat
...
11 828 665 001
Principle
A single reagent lyses the sample and inactivates RNase
...
The RNA remains bound while a DNase treatment and a series
of rapid “wash-and-spin” steps remove DNA and contaminating small molecules
...
The process does
not require precipitation, organic solvent extractions, or extensive handling of the RNA
...
0)
109 gram positive or gram negative bacteria
Application
Preparation of intact total RNA, which may be used directly in RT-PCR, Differential
Display RT-PCR (DDRT-PCR), Northern blotting, primer extension assays, RACE
(rapid amplification of cDNA ends), cDNA library construction, in vitro translation,
or nuclease protection assays
...
Time required
Total time: approx
...
Purity: Purified RNA is free of DNA, protein, salts, and other cellular components
...
deal for a wide variety of samples, because one kit can purify RNA from many
I
sources
...
Increases lab safety, because the kit does not use hazardous organic solvents
...
Flow diagram
Harvest 106 cultured
cells and resuspend
cell pellet
2
Add 200 µl PBS
Add 400 µl Lysis/ Binding
Buffer
Mix well and pipette the
sample in the upper
reservoir of the High Pure
Filter Tube assembly
Centrifuge for 15 s
at 8000 x g
For each sample add
mixture of 90 µl DNase I
Incubation Buffer and
10 µl DNase I
Discard flowthrough
Incubate for 15 min
at +15 to +25°C
Add 500 µl Wash Buffer I
Centrifuge for 15 s
at 8000 x g
Add 500 µl Wash Buffer II
Discard flowthrough
Centrifuge for 15 s
at 8000 x g
Add 200 µl Wash Buffer II
Discard flowthrough
Centrifuge for 2 min
at max
...
Kit contents
Lysis/Binding Buffer containing guanidine HCl and Triton X-100 (25 ml)
DNase I, lyophilized (10 kU)
Dissolve DNase I in 0
...
A
0
...
DNase Incubation Buffer (10 ml)
Wash Buffer I containing guanidine HCl (33 ml)
Add 20 ml absolute ethanol to Wash Buffer I before use
...
Elution Buffer (30 ml)
High Pure Filter Tubes (50 tubes)
Collection Tubes, 2 ml (50 tubes)
III
...
5 ml, sterile
Phosphate buffered saline (PBS), sterile
Red Blood Cell Lysis Buffer (for human blood)
2
Lysozyme, 50 mg/ml (for bacteria)
10 mM Tris-HCl, pH 8
...
5 mg/ml (for yeast)
Standard tabletop centrifuge capable of 3000 x g centrifugal force (for yeast or
bacteria)
IV
...
cerevisiae)
108 cells
20
Bacteria (E
...
subtilis)
10 cells
35
High Pure Kits and Silica Adsorption
6
9
9
57
High Pure RNA Isolation Kit
How to use the kit
V
...
For details, see
the package insert provided with the kit
...
Isolation of total RNA from 106 cultured mammalian cells
2
This protocol may also be used to isolate RNA from small blood volumes (<200 µl),
as an alternative to Protocol Vb
...
Add 400 µl Lysis/Binding Buffer to the resuspended cells
...
To transfer the sample to a High Pure Tube:
E Insert one High Pure Filter Tube into one Collection Tube
...
700 µl)
...
8000 x g
...
E Reinsert the Filter Tube in the same Collection Tube
...
Then:
E Add 100 µl DNase solution to the upper reservoir of the Filter Tube
...
After the DNase incubation:
E Add 500 µl Wash Buffer I to the upper reservoir of the Filter Tube
...
After the first wash:
E Add 500 µl Wash Buffer II to the upper reservoir of the Filter Tube
...
After the second wash:
E Add 200 µl Wash Buffer II to the upper reservoir of the Filter Tube
...
13,000 x g) to
remove any residual Wash Buffer
...
5 ml
microcentrifuge tube
...
E Centrifuge the tube assembly for 1 min at 8000 x g
...
58
Nucleic Acid Isolation and Purification Manual
High Pure RNA Isolation Kit
How to use the kit
Vb
...
It should have been collected in a
tube containing sodium EDTA or any other anticoagulant
...
Prepare sample tubes:
E Warm Red Blood Cell Lysis Buffer to +15 to +25°C
...
5 ml microcentrifuge tube
...
E Cap and mix by inversion
...
Mix the tubes by:
E EITHER placing the microcentrifuge tube on a rocking platform or gyratory shaker
for 10 min
...
Centrifuge the tube at 500 x g for 5 min in a microcentrifuge
...
For each tube:
E Add 1 ml Red Blood Cell Lysis Buffer to the white pellet, then cap the tube
...
Do not vortex the tube
...
With a sterile pipette, carefully remove and discard both the supernatant and the red
ring of blood cell debris that forms around the outer surface of the white blood cell
pellet
...
Thus:
E Resuspend the white pellet in 200 µl sterile PBS
...
Alternatively blood stabilized with the RNA/DNA Stabilization Reagent for Blood/
Bone Marrow can be used
Mix 0
...
E This mixture is stable for at least one week at +2 to +8°C or a month at
–15 to –25°C
...
High Pure Kits and Silica Adsorption
59
High Pure RNA Isolation Kit
How to use the kit
Follow protocol Va starting with the DNase incubation (Step 5)
...
Isolation of total RNA from 108 yeast (S
...
0)
...
E Resuspend the pellet in 200 µl of PBS and transfer the suspension to a sterile 1
...
Add 10 µl lyticase solution (0
...
Incubate the
tube for 15 min at +30°C
...
Vd
...
E Resuspend the pellet in 200 µl of 10 mM Tris-HCl, pH 8
...
5 ml microcentrifuge tube
...
0) to each microcentrifuge
tube
...
Add 400 µl Lysis/Binding Buffer to the tube and mix well
...
E Pipette entire sample into upper buffer reservoir of the Filter Tube
...
Prepare DNase solution as in Step 5, Protocol Va
...
E Incubate the Filter Tube for 60 min at +15 to +25°C
...
VI
...
For details on
how to troubleshoot the above protocols, see the General Troubleshooting Procedure for
all High Pure kits on page 113 of this manual
...
60
Nucleic Acid Isolation and Purification Manual
High Pure RNA Isolation Kit
Typical results with the kit
Typical results with the kit
Experiment 1
RT-PCR
+DNase I
+DNase I
–DNase I
PCR
–DNase I
2
1
2
3
4
5
6
7
1
2
3
4
5
6
7
F
igure 12: Effect of DNase treatment on mammalian RT-PCR templates prepared with the High Pure
RNA Isolation Kit
...
Four samples were treated with the DNase solution as described in Protocol Va above
...
10 µl of each sample was used in a first strand cDNA reaction (20 µl total volume) with
M-MuLV Reverse Transcriptase and oligo(dT) (as primer)
...
Left panel:
Lane 1: MWM VI
Lanes 2 – 7: The RT-PCR produced a specific 983 bp amplification product
...
Result: The samples not incubated with DNase gave an amplification product in the PCR control, indicating the
presence of some residual genomic DNA (right panel, lanes 6 and 7), samples that were treated with DNase did
not give any amplification product (right panel, lanes 2 – 5) in the control
...
3 µg 1
...
0 µg 0
...
0 µg 3
...
RNA was isolated from 106 K562 human lymphocyte
cells with each kit, and the yields determined spectrophotometrically
...
The blot was hybridized with a digoxigeninlabeled actin RNA probe (Roche Applied Science)
...
Result: A specific signal of the expected size was
obtained in all samples, but the specific actin RNA bands
in the aliquots prepared with the Roche Applied Science
kit were more concentrated and contained less background
...
CT
CT
Figure 14: Mammaglobin
Figure 15: PBGD
2
PBGD
62
Figure 16: High Pure total RNA
Preparation from human whole blood
(research samples)
...
10 µl Blood = 30 µl Lysate
20 µl Blood = 60 µl Lysate
50 µl Blood = 150 µl Lysate
100 µl Blood = 300 µl Lysate
200 µl Blood = 600 µl Lysate
300 µl Blood = 900 µl Lysate
Nucleic Acid Isolation and Purification Manual
High Pure RNA Isolation Kit
Reference
Anz D
...
(2010) J
...
, 184, 939 – 946
...
et al
...
Acids Res
...
del Fresno C
...
(2009) J
...
, 182, 6494 – 6507
Handke W
...
(2009) J
...
, 182, 2849 – 2858
...
et al
...
et al
...
Biol
...
, 284, 10514 – 10522
Kouwen T
...
R
...
et al
...
Envir
...
, 76, 659 – 669
Majchrzykiewicz J
...
et al
...
Agents Chemother
...
et al
...
Immunol
...
et al
...
No
...
Frequently
applied techniques like Reverse Transcriptase-PCR (RT-PCR), Northern blotting, and
RNase protection require the use of intact undegraded RNA
...
After adding ethanol RNA binds selectively to a glass fiber fleece in the presence of
a chaotropic salt (guanidine HCI)
...
During a series of rapid ”wash-and-spin” steps
to remove contaminating cellular components the RNA remains bound to the glass fiber
fleece
...
The process
does not require RNA precipitation, organic solvent extractions, or extensive handling of
the RNA
...
g
...
Time required
Total time: approx
...
Add additional time
when using alternative disruption methods
...
3 – 3 µg total RNA per mg tissue depending on tissue type
...
The absence of contaminating DNA is examined by a PCR without a preceding RTreaction; no amplification product is obtained
...
Eluates are ready-to-use, no alcohol precipitation has to be done
...
Flow diagram
Lysate supernatant
(tissue homogenized in
Lysis/Binding Buffer)
Mix and apply lysate
(max
...
5 volumes ethanol
abs
...
Kit contents
Lysis/Binding Buffer (25 ml)
DNase I, lyophilizate (10 kU)
DNase Incubation Buffer (10 ml)
Wash Buffer I (33 ml)
Add 20 ml absolute ethanol before use
Wash Buffer II (10 ml)
Add 40 ml absolute ethanol before use
Elution Buffer (30 ml)
High Pure Filter Tubes, (50 tubes)
Collection Tubes, 2 ml (50 tubes)
High Pure Kits and Silica Adsorption
65
High Pure RNA Tissue Kit
How to use the kit
III
...
5 ml, sterile
Mortar and pestle or Rotor-Stator Homogenizer (e
...
, Ultra Turrax)
2
IV
...
The yield with mouse
muscle tissue could not be determined spectroscopically, but isolated RNA resulted in a
specific RT-PCR signal
...
5 – 2
...
5 – 1
...
5 – 3
...
3 – 0
...
3
muscle
n
...
V
...
Standard reaction
Depending on the disruption and homogenization method, add one of the following to
a nuclease-free 1
...
20 – 25 mg); disrupt and homogenize the tissue using a rotor-stator homogenizer
...
For optimal yield do not exceed 10 mg tissue
...
Add 200 µl absolute ethanol to the lysate supernatant and mix well
...
Centrifuge for 30 s at maximum speed (13,000 x g) in a standard table top microcentrifuge
...
Discard the flowthrough and reassemble the Filter Tube and the used Collection Tube
...
5 ml reaction tube, pipette 90 µl DNase Incubation Buffer, add 10 µl
DNase I working solution, mix
...
Incubate for 15 min at +15 to +25°C
...
E Discard the flowthrough and again reassemble the Filter Tube and the used
Collection Tube
...
Discard the flowthrough and again reassemble the Filter Tube and the used Collection
Tube
...
13,000 x g) to remove residual Wash Buffer
...
2
Residual ethanol may interfere with subsequent reactions
...
E Insert the Filter Tube in a nuclease-free 1
...
Add 100 µl Elution Buffer to the upper reservoir of the Filter Tube
...
The microcentrifuge tube now contains the eluted RNA
...
VI
...
For details on
how to troubleshoot the above protocol, see the General Troubleshooting Procedure for
all High Pure Kits on page 113 of this manual
...
Typical results with the kit
Comparison of different lysis/homogenization procedures
Experiment 1
1
2
High Pure Kits and Silica Adsorption
3
4
5
6
7
Figure 17: Mouse liver was homogenized with
various procedures and RNA purified using the
standard protocol of the High Pure RNA Tissue
Kit
...
Lane 1: Ultra Turrax;
Yield: 1
...
0
Lane 2: Disposable plastic pestle, motor driven;
Yield: 3
...
0
Lane 3: Mortar + pestle/20G needle;
Yield: 1
...
0
Lane 4: Disposable plastic pestle, manual; 20 G
needle;
Yield: 1
...
0
Lane 5: Disposable plastic pestle, manual;
Yield: 3
...
0
Lane 6: Bead-Vortex homogenization;
Yield: 3
...
0
Lane 7: MWM
67
High Pure RNA Tissue Kit
Experiment 2
A
Platinum-resistant ovarian cancer samples
Platinum-sensitive ovarian cancer samples
Sample
RNA-Integrity-Number
Sample
RNA-Integrity-Number
1
8
...
7
2
12
8
...
2
13
8
...
7
14
8
...
5
15
9
...
2
16
9
...
8
17
8
...
1
18
8
...
1
19
9
...
0
3
9
...
4
B
C
Figure 18: Measurement of RNA quality ranking using the Agilent Bioanalyzer results in a RNA
Integrity Number (RIN) between 10 (highest) and 1 (lowest)
...
Panels B and C show an electropherogram overlay of several platinum-resistant (B) and platinum-sensitive (C)
ovarian tumor samples
...
Reference
Ethier-Chiasson M
...
(2008) Reproduction, 136, 491 – 502
Geletneky K
...
(2010) Neuro Oncology, 12: 804 - 814
Keating P
...
(2009) Immunol
...
, 87, 534 – 545
Li Y
...
(2008) J
...
Soc
...
, 19, 252 – 259
Nakamura, S
...
(2007) Nephrol
...
Transplant
...
et al
...
, 35, 2229 – 2235
Rivera R
...
et al
...
Mol
...
, 17, 1 – 14
Rocks N
...
(2008) Cancer Res
...
et al
...
Biol
...
, 285: 2184 - 2192
Terahara K
...
(2008) J
...
, 180, 7840 – 7846
68
Nucleic Acid Isolation and Purification Manual
High Pure FFPE RNA Micro Kit
High Pure FFPE RNA Micro Kit
for isolation of total RNA from formalin-fixed, paraffin-embedded tissue samples
Cat
...
04 823 125 001 (up to 50 isolations)
Principle
Starting material
To prepare tissue sections for RNA isolation, fixation reagents must be removed from the
samples; after deparaffinization, the sections are ready to be processed with the High
Pure FFPE RNA Micro Kit
...
The homogenate is then applied to the glass fiber
fleece in a High Pure Micro Filter Tube
...
DNA is removed from the filter by
digestion with DNase I
...
Finally, the purified RNA is
eluted from the column in low-salt buffer
...
g
...
human research samples)
Section thickness as well as yield and quality of the isolated RNA are strongly related
to type of tissue, age of sample as well as fixation protocol used
...
The quality of
RNA from paraffin-sections achieved with the kit is suitable for the relative quantification of mRNA with RT-PCR especially on the LightCycler® 2
...
Time required
Total time required is approx
...
Average yields are 1
...
5 µg /
5 µm section DNA-free RNA
...
Obtain a highly concentrated eluate (10 µl) and recovery (>80%)
...
Efficiently isolate RNA – even small RNA fragments
...
Rely on the innovative column design to efficiently recover contamination-free
RNA
...
Flow diagram
Place deparaffinized
section in a reaction tube
Add 60 µl Tissue Lysis Buffer
and 10 µl 10% SDS
2
Vortex 3 x 4 s and spin down
Add 30 µl Proteinase K
Vortex 3 x 4 s, spin down and
incubate for 3 h at +55°C
Add 200 µl Binding Buffer
and 200 µl ethanol, abs
...
Centrifuge at 8,000 x g for 30 s
Discard flowthrough
Centrifuge 1 min at max
...
speed to dry filter
...
5 ml microcentrifuge tube
...
Centrifuge at
8,000 x g for 1 min
Reload the Eluate, incubate for
1 min at +15 to +25°C
...
Kit contents
Tissue Lysis Buffer, 20 ml
Proteinase K (recombinant, PCR grade), lyoph
...
, 4 kU
DNase Incubation Buffer, 10x conc
...
Additional materials needed
Absolute ethanol
Ethanol 70%
Hemo-De or Xylene
SDS 10%
Microcentrifuge tubes, 1
...
0 ml, sterile
Standard tabletop microcentrifuge capable of 13,000 x g centrifugal force
IV
...
Deparaffinization procedure for one 1-10 µm section of formalinfixed, paraffin-embedded tissue in a 1
...
1
To one 1 – 10 µm section in a 1
...
Centrifuge for 2 min at maximum speed
(12,000 – 14,000 x g) and discard supernatant by aspiration
...
If the tissue appears to be floating in the Xylene, spin for a further 2 min
...
3
Add 800 µl ethanol abs
...
Centrifuge for 2 min at maximum speed (12,000 – 14,000 x g) and discard supernatant
by aspiration
...
Centrifuge for 2 min at maximum speed and discard supernatant by aspiration
...
Incubate the open tubes in a heating block for 5 – 15 min at +55°C to air dry the
tissue pellet
...
Deparaffinization procedure for one 1 – 10 µm section of
f
ormalin-fixed, paraffin-embedded tissue on a microscope slide
...
for 10 min
...
4
2
Place the slide in a Hemo-De (or Xylene) bath and incubate for 10 min
...
5 ml reaction tube
...
5
E Dry the tissue for 10 min at +55°C
...
IVc
...
If necessary, multiple preparations can be pooled after step 4
...
E Vortex 3 x 4 s, spin down and incubate for 3 h at +55°C
...
E Vortex 3 x 4 s and spin down
3
Combine the High Pure filter tube with a collection tube and pipette the lysate into the
upper reservoir
...
Steps 3-4 can be repeated, in order to load the column with additional sample
material (do not overload the column)
...
speed to dry filter
...
7
E Add 300 µl Wash Buffer I working solution to the upper reservoir
...
8
E Add 300 µl Wash Buffer II working solution
E Centrifuge for 15 s at 8000 x g, discard the flowthrough
...
Place the High Pure Micro filter tube in a fresh collection tube and centrifuge for
2 min at maximum speed
...
5 ml reaction tube, add 20 µl
Elution Buffer and incubate for 1 min at +15 to +25°C
...
E Reload the eluate; incubate for 1 min at +15 to +25°C
...
Either use 0
...
Before photometric determination of the RNA concentration, centrifuge the
eluate for 2 min at maximum speed and transfer supernatant to a fresh 1
...
V
...
For details on
how to troubleshoot the above protocols, see the General Troubleshooting Procedure for
all High Pure kits on page 113 of this manual
...
2
Typical results with the kit
Marker Peak
High Pure FFPE RNA Micro Kit
Supplier X
Figure 19: Fragment-length distribution of
isolated RNA
...
The size distribution of the
recovered RNA fragments was determined on
an electropherogram (Bioanalyzer, Agilent)
...
HP FFPE RNA Micro
(Slope -3
...
107)
Figure 20: Performance of isolated RNA in RT-PCR
...
The isolated RNA samples were serially diluted and used as templates in separate RT-PCRs
...
5 Instrument and LightCycler®
RNA Amplification Kit SYBR Green I
...
High Pure Kits and Silica Adsorption
73
High Pure FFPE RNA Micro Kit
References
Boniface K
...
(2010) J
...
, 185: 679 - 687
Evtimova, V
...
(2006) Biochemica 1, 16 – 17
Humar B
...
(2009) Cancer Res
...
et al
...
et al
...
Biol
...
M
...
(2009) J
...
, 183, 4693 – 4704
Steinmetz O
...
et al
...
, 74, 448 – 45
Nucleic Acid Isolation and Purification Manual
High Pure RNA Paraffin Kit
High Pure RNA Paraffin Kit
for up to 100 isolations of total RNA from fresh-frozen and formalin-fixed, paraffinembedded tissue
Cat
...
03 270 289 001
Principle
Tissue samples are disrupted and homogenized during an overnight incubation with
Proteinase K (paraffin samples) or by using a suitable tissue homogenizer (fresh-frozen
tissue)
...
The binding process is specific for
nucleic acids in general, but the binding conditions are optimized for RNA
...
Residual DNA is digested by incubation with DNase I
...
Finally, low salt elution removes the
nucleic acids from the glass fiber
...
Starting material
2
– 13 µg sections from formalin-fixed, paraffin-embedded tissue (research
10
samples)
10 – 30 mg fresh-frozen solid tissue (research samples)
3 – 5 µm tissue section from fresh-frozen tissue (research samples)
Application
The kit is designed for the isolation of total RNA for use in RT-PCR
...
0 Instrument
...
2 h without overnight incubation
Results
– 1
...
3
depending on tissue type and section size
...
2
– 5 µg total RNA per 3 x 5 µg section from fresh solid tissue depending on tissue
0
...
Benefits
Toxic materials are avoided, no organic solvent extraction is necessary
...
Improved purity due to extensive purification method
...
Flow diagram
Place deparaffinized
section in a reaction tube
2
Vortex briefly in several
intervals and incubate
overnight at +55°C
Add 100 µl Tissue Lysis
Buffer, 16 µl 10% SDS and
40 µl Proteinase K
Add 325 µl Binding Buffer
and 325 µl ethanol, abs
...
Repeat the
step for remaining lysate
Centrifuge for 30 s at
8000 x g
...
speed to dry filter fleece
Discard flowthrough
Discard flowthrough
Discard flowthrough
Discard flowthrough
Discard Collection Tube
Add 500 µl Wash Buffer I
Centrifuge for 15 s
at 8000 x g
Add 500 µl Wash Buffer II
Centrifuge for 15 s
at 8000 x g
Add 300 µl Wash Buffer II
Centrifuge for 15 s
at 8000 x g
Centrifuge for 2 min
at max
...
Place Filter Tube in fresh
1
...
Centrifuge 1 min at
8000 x g
Add 90 µl Elution Buffer
Add 10 µl DNase Incubation
Buffer and 1
...
Mix and pipette into the
upper reservoir of a
High Pure Filter Tube
assembly and centrifuge
for 30 s at 8000 x g
Discard flowthrough
Repeat centrifugation at
max
...
speed
Add 50 µl Elution Buffer
Place Filter Tube in fresh
1
...
Kit contents
Tissue Lysis Buffer (20 ml)
Proteinase K (2 x 100 mg)
Binding Buffer (2 x 80 ml)
Wash Buffer I (100 ml)
Add 60 ml absolute ethanol before first use
Wash Buffer II (50 ml)
Add 200 ml absolute ethanol before first use
DNase I (4 kU)
DNase Incubation Buffer, 10 x conc
...
Additional materials needed
2
Hemo-De or Xylene
Ethanol, absolute
SDS 10%
Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force
Microcentrifuge tubes, sterile
Homogenization device
IV
...
Different fixation procedures of the tissue
may lead to variations regarding RNA yields
...
µl of the RNA eluate is used in one-step RT-PCR and specific primers for b2M10
gene
...
Absence of contaminating genomic DNA is examined by a RT-PCR without addition
of reverse transcriptase; no amplification product is obtained
...
Isolation procedure
Va
...
To one tissue pellet (deparaffinized as described in package insert) add 100 µl Tissue
Lysis Buffer, 16 µl 10% SDS and 40 µl Proteinase K
...
Add 325 µl Binding Buffer and 325 µl ethanol abs
...
Combine the High Pure Filter Tube and the Collection Tube and pipette the lysate into
the upper reservoir
...
Step 3 – 4 can be repeated, in case RNA needs to be pooled, with 2 more tissue
pellet preparations
...
Add 500 µl Wash Buffer I working solution to the upper reservoir
...
Add 500 µl Wash Buffer II
...
Add 300 µl Wash Buffer II
...
Centrifuge the High Pure Filter Tube for 2 min at maximum speed
...
5 ml reaction tube, add 90 µl Elution Buffer
...
Add 10 µl DNase Incubation Buffer, 10x and 1
...
Incubate for 45 min at +37°C
...
Vortex briefly
...
Add 325 µl Binding Buffer and 325 µl ethanol abs
...
Centrifuge for 30 s at 8000 x g in a microcentrifuge and discard the flowthrough
...
Add 500 µl Wash Buffer I to the upper reservoir
...
Add 500 µl Wash Buffer II
...
Add 300 µl Wash Buffer II
...
Centrifuge the High Pure Filter Tube for 2 min at maximum speed
...
5 ml reaction tube
...
Centrifuge for 1 min at 8000 x g to collect
the eluted RNA
...
High Pure Kits and Silica Adsorption
79
High Pure RNA Paraffin Kit
How to use the kit
Vb
...
Centrifuge the lysate for 2 min at maximum speed in a microcentrifuge
...
Add 1 ml Binding Buffer and 600 µl ethanol abs
...
Combine the High Pure Filter Tube and the Collection Tube and pipette half of the
volume of the lysate into the upper reservoir
...
Pipette the rest of the lysate into the same High Pure Filter Tube and centrifuge for
30 s as described in step 5
...
Add 500 µl Wash Buffer I to the upper reservoir
...
Add 500 µl Wash Buffer II
...
Add 300 µl Wash Buffer II
...
Centrifuge for 2 min at maximum speed
...
5 ml reaction tube
...
Centrifuge for 1 min at 8000 x g
...
0 µl DNase I working solution to the eluate
and mix
...
Add 20 µl Tissue Lysis Buffer 18 µl 10% SDS and 40 µl Proteinase K
...
Incubate for 1 h at +55°C
...
, mix by pipetting
...
Centrifuge for 30 s at 8000 x g in a microcentrifuge and discard the flowthrough
...
Add 500 µl Wash Buffer I to the upper reservoir
...
Add 500 µl Wash Buffer II
...
Add 300 µl Wash Buffer II
...
u
Centrifuge the High Pure Filter Tube for 2 min at maximum speed
...
5 ml reaction tube
...
Centrifuge for 1 min at 8000 x g to collect the eluted RNA
...
The complete
disruption of cell walls and plasma membranes of cells and organelles is absolutely
required to release all the RNA contained in the sample
...
Homogenization is necessary to reduce the
viscosity of the cell lysates produced by disruption
...
Incomplete homogenization results in significantly
reduced yields
...
80
Nucleic Acid Isolation and Purification Manual
High Pure RNA Paraffin Kit
How to use the kit
Vc
...
5 ml reaction tube add 100 µl Tissue
Lysis Buffer 16 µl 10% SDS and 40 µl Proteinase K
...
Add 325 µl Binding Buffer and 325 µl ethanol abs
...
Combine the High Pure Filter Tube and the Collection Tube and pipette the lysate into
the upper reservoir
...
Repeat the centrifugation at maximum speed in order to dry the filter fleece completely
...
Centrifuge for 15 s at 8000 x g, discard
the flowthrough
...
Centrifuge for 15 s at 8000 x g, discard the flowthrough
...
Centrifuge for 15 s at 8000 x g, discard the flowthrough
...
Place the High Pure Filter Tube in a fresh 1
...
Add 90 µl Elution
Buffer
...
Add 10 µl DNase Incubation Buffer, 1
...
Incubate for 45 min at +37°C
...
Vortex briefly
...
Add 325 µl Binding Buffer and 325 µl ethanol abs
...
Centrifuge for 30 s at 8000 x g in a microcentrifuge and discard the flowthrough
...
Add 500 µl Wash Buffer I to the upper reservoir
...
Add 500 µl Wash Buffer II
...
Add 300 µl Wash Buffer II
...
Centrifuge the High Pure Filter Tube for 2 min at maximum speed
...
5 ml reaction tube
...
Centrifuge for 1 min at 8000 x g to collect the eluted RNA
...
2
VI
...
For details on
how to troubleshoot the above protocol, see the General Troubleshooting Procedure for
all High Pure kits on page 113 of this manual
...
High Pure Kits and Silica Adsorption
81
High Pure RNA Paraffin Kit
Typical results with the kit
Typical results with the kit
Experiment 1
2
Figure 21: Linear measuring range of TP, DPD TS and G6PDH
LightCycler® System of RNA dilution series for TP, DPD, TS and G6PDH (1
...
5 ng RNA)
...
Each RNA
concentration was 5 times in an independent reaction reverse transcripted, using specific primers
...
The determined dynamic range
covers 2 log stages of the analyzed RNA concentrations
...
RT controls for each target were performed and negative in subsequent LC PCR assays (data not
shown)
...
C
...
et al
...
Natl
...
, 101, 736 – 750
Evtimova, V
...
(2006) Biochemica 1
Hamatani K
...
(2008) Cancer Res
...
et al
...
, 69, 7385 – 7392
How A
...
(2010) Invest
...
Vis
...
, 51, 928 – 932
Lee H
...
et al
...
Immunol
...
et al
...
-M
...
(2009) Clin
...
, 15, 731 – 739
Volante M
...
(2009) J
...
Endocrinol
...
, 94, 4735 – 4741
High Pure Kits and Silica Adsorption
2
83
High Pure miRNA Isolation Kit
High Pure miRNA Isolation Kit
for low to medium miRNA isolation
Cat
...
05 080 576 001 (up to 50 miRNA isolations)
In the presence of the chaotropic salt guanidine thiocyanate, RNA binds selectively to
special glass fibers pre-packed in the High Pure Filter Tube
...
This simple method
eliminates the need for organic solvent extractions and RNA precipitation, allowing for
rapid purification of many samples simultaneously
...
When the concentration of
Binding enhancer is increased the small RNA fraction can be bound to a second High
Pure Filter Tube
...
g
...
30 min (without Proteinase K incubation and deparaffinization in the preparation of paraffin-embedded tissue samples)
...
Purity: Purified RNA is free of DNA, protein, salts and other cellular components
...
g
...
It can be used
to purify total RNA or to prepare samples enriched for small RNA < 100 nucleotides
...
g
...
Eliminate the need for hazardous organic solvents
...
Obtain high yields with a simple, efficient protocol
...
Choose one flexible kit for all your miRNA purifications
...
Nucleic Acid Isolation and Purification Manual
High Pure miRNA Isolation Kit
How to use the kit
How to use the kit
I
...
700 µl at a time) to a
High Pure Filter Tube
assembly, centrifuge at
13,000 × g for 30 - 60 s
(repeat if lysate volume
is more than 700 µl)
...
5 ml
microcentrifuge tube
Add 50 - 100 µl Elution
Buffer on the center
of the filter
Incubate for 1 min
at +15 to +25°C
...
700 µl at a time) to a
High Pure Filter Tube
assembly, centrifuge at
13,000 × g for 30 – 60 s
(repeat if lysate volume
is more than 700 µl)
...
Add 500 µl Wash Buffer
Centrifuge at 13,000 × g
for 30 s
Add 300 µl Wash Buffer
Disc ard flowthrough
Centrifuge at 13,000 × g
for 30 s
Disc ard flowthrough
Centrifuge at 13,000 × g
for 1 min
Discard collection tube
Place the Filter Tube in a
fresh 1
...
Centrifuge at 13,000 × g
for 1 min
Purified small RNA
86
Nucleic Acid Isolation and Purification Manual
High Pure miRNA Isolation Kit
How to use the kit
II
...
If any solution contains a precipitate, warm the solution prior
to use at room temperature or in a +37°C water bath to dissolve the precipitate
...
Additional materials needed
Absolute ethanol
10 % SDS solution (for FFPE sections only)
Hemo-De or Xylene (for FFPE sections only)
Standard tabletop microcentrifuge capable of 13,000 x g centrifugal force
Microcentrifuge tubes, 1
...
0 ml, sterile
Mortar and Pestle, MagNA Lyser Instrument or Rotor-Stator Homogenization device
(e
...
, Ultra Turrax)
IV
...
5 — 9
Mouse kidney
0
...
5 — 8
Rat brain
0
...
5 — 3
...
5 — 2
...
Protocols for preparing total RNA including miRNA or
enriched miRNA
Va
...
E Add 312 µl Binding Buffer, vortex briefly, then add 200 µl Binding Enhancer
...
3
Centrifuge for 30 s at 13,000 × g, discard the flowthrough
...
4
Add 500 µl Wash Buffer working solution
...
5
After first wash:
E Add 300 µl Wash Buffer working solution
...
6
To dry the glass fiber fleece:
E Centrifuge at 13,000 × g for 1 min
...
5 ml microcentrifuge tube,
E Add 100 µl Elution Buffer and incubate for 1 min at +15 to +25°C
...
An elution step with 100 µl Elution Buffer will increase
the total yield by approx
...
E Centrifuge for 1 min at approx
...
The microcentrifuge tube now contains the eluted total RNA
...
5 – 5 µl of
the eluted RNA directly in RT-PCR or store the eluted RNA at –80°C for later analysis
...
Before determining the RNA concentration photometrically, centrifuge and
transfer supernatant to a fresh 1
...
EEE
88
Nucleic Acid Isolation and Purification Manual
High Pure miRNA Isolation Kit
How to use the kit
Two-Column Protocol (purified, enriched small RNA)
To prepare the sample, binding buffer mixture
E Take 150 µl of cell lysate
For optimum results do not add lysate from more than 10 mg of animal
tissue, 50 mg of plant tissue, or 106 animal or plant cells to the column at
this step
...
2
E Vortex 3 × 5 s
2
To transfer the sample to a High Pure Tube
E Combine the High Pure Filter Tube with a collection tube and pipette the whole
mixture from step 1 into the upper reservoir
...
To minimize pipetting steps a 2 ml microcentrifuge tube can be used instead of
the collection tube
...
E Vortex 3 × 5 s
...
5
Centrifuge for 30 s at 13,000 × g, discard the flowthrough
...
6
Prepare the first wash
E Add 500 µl Wash Buffer working solution
...
7
After the first wash:
E Add 300 µl Wash Buffer working solution
...
8
To dry the glass fiber fleece:
E Centrifuge at 13,000 × g for 1 min
...
5 ml microcentrifuge tube, add
100 µl Elution Buffer and incubate for 1 min at +15 to +25°C
...
An elution step with 100 µl Elution Buffer will increase the
total yield by approx
...
E Centrifuge for 1 min at 13,000 × g
...
Either use
0
...
High Pure Kits and Silica Adsorption
89
High Pure miRNA Isolation Kit
How to use the kit
Vb
...
For a deparaffinization protocol of
FFPE slices please review the package insert
...
2
To increase yield, incubate at +55°C for at least 3 h (up to overnight)
...
2
To transfer the sample to a High Pure Tube
E Combine the High Pure Filter Tube with a 2 ml collection tube and pipette the
lysate into the upper reservoir
...
E Centrifuge for 30 s at 13,000 × g in a microcentrifuge and
3
Collect the flowthrough
...
4
Combine the High Pure Filter Tube with a collection tube and pipette the whole
mixture from step 6 into the upper reservoir
...
Steps 2 - 3 can be repeated in order to load the column with additional sample material (do not overload the column)
...
E Centrifuge for 30 s at 13,000 × g, discard the flowthrough
...
E Centrifuge for 30 s at 13,000 × g, discard the flowthrough
...
8
To elute the RNA:
E Place the High Pure Filter Tube in a fresh 1
...
E Add 100 µl Elution Buffer and incubate for 1 min at +15 to +25°C
...
E Centrifuge for 1 min at 13,000 × g
...
Either use
0
...
90
Nucleic Acid Isolation and Purification Manual
High Pure miRNA Isolation Kit
How to use the kit
Vc
...
1
Preparation of DNase Solution:
E For 70 µl DNase solution mix 49
...
5 µl 10 × DNase
Incubation buffer and 1 µl DNase 1 (10 U/µl)
2
DNase treatment on the column is not recommended as miRNA will elute
at low salt concentrations
...
100 µl eluate add 56 µl DNase Solution
...
E Incubate for 30 min at +37°C
...
E Vortex briefly
...
4
Combine the High Pure Filter Tube with a 2 ml collection tube and pipette the DNase
treated sample into the upper reservoir
...
5
Prepare the first wash:
E Add 500 µl Wash Buffer working solution
...
6
After the first wash:
E Add 300 µl Wash Buffer working solution
...
7
To dry the glass fiber fleece:
E Centrifuge at 13,000 × g for 1 min, in order to dry the filter fleece completely
...
5 ml microcentrifuge tube, add 100 µl
Elution Buffer and incubate for 1 min at +15 to +25°C
...
An elution step with 100 µl Elution Buffer will increase
the total yield by approx
...
E Centrifuge for 1 min at 13,000 × g
...
Either use
0
...
Glass fibers in the eluate may interfere with optical density measurement
...
5 ml reaction tube
...
Troubleshooting the High Pure Protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
High Pure Kits and Silica Adsorption
91
High Pure miRNA Isolation Kit
Typical results with the kit
Typical results with the kit
Experiment 1
Eluate 1
total RNA
Eluate 2
Eluate 3
Purified small RNA
2
5
...
A 1 µg sample of miRNA 145 was spiked into a liver tissue lysate before
purification
...
Alternatively, electropherograms were recorded on an Agilent Bioanalyzer (Fig 25)
...
The absence of contaminating DNA is examined by PCR without a
preceding RT-reaction; no amplification product is obtained (data not shown)
...
Half of the organs were fresh-frozen,
and the other half formalin-fixed and paraffin-embedded
...
Equal amounts (10 μm sections) of FFPE tissue were used for
comparison of the High Pure miRNA Isolation Kit with 2 kits from alternative suppliers
...
Isolated total RNA from each preparation was quantified on a
Nanodrop instrument
...
Reference
Abe M
...
(2010) Plant Physiology, 154: 1335 - 1346
Santosh K et al
...
, 70, 36 – 45
Høiby P
...
et a
...
No
...
Then, in the presence of a chaotropic salt (guanidine HCl), viral RNA binds selectively to glass fiber fleece in a special
centrifuge tube
...
Finally, low salt elution removes the NA
from the glass fiber fleece
...
Starting material
Research samples (200 – 600 µl) may be:
Serum
2
Plasma
Tears
ell culture supernatant
C
Urine
Breast milk
Application
Preparation of intact viral genomic RNA, which may be used directly as templates for
RT-PCR
...
10 min
Hands-on time: <10 min
Results
Yield: 50 µl eluate is enough for 8 – 14 RT-PCRs
...
Benefits
Saves time, because the kit can prepare multiple RT-PCR templates in just minutes
...
Minimizes RNA loss, because the kit removes contaminants without time-consuming precipitation or solvent extraction
...
High Pure Kits and Silica Adsorption
95
High Pure Viral RNA Kit
How to use the kit
How to use the kit
I
...
speed
(13,000 x g) for 10 s
Discard flowthrough and
Collection Tube
Add 50 µl Elution Buffer
Centrifuge at 8000 x g
for 1 min
Purified Viral RNA
II
...
Wash Buffer (2 x 10 ml)
Add 40 ml absolute ethanol to each Wash Buffer before use
...
Additional materials needed
Absolute ethanol
Standard tabletop microcentrifuge capable of a 13,000 x g centrifugal force
Microcentrifuge tubes, 1
...
Protocol for preparing RNA from 200 µl samples
See the package insert supplied with the kit for instructions on processing
200 – 600 µl samples
...
5 ml microcentrifuge tube:
E Add 200 µl serum or plasma
...
2
The RNA yield can be increased two-fold with an optional incubation step,
thus resulting in higher sensitivity
...
This
incubation step can be omitted when time to result is critical
...
E Pipette entire sample into upper buffer reservoir of the Filter Tube
...
8000 x g
...
E Insert the Filter Tube into a new Collection Tube
...
E Discard flowthrough and combine Filter Tube with a new Collection Tube
...
E Repeat the centrifugation (as in step 3)
...
E Repeat the wash step (Step 6)
...
13,000 x g) to remove residual Wash Buffer
...
5 ml
microcentrifuge tube
...
E Centrifuge the tube assembly for 1 min at 8000 x g
...
You may:
E EITHER use an aliquot of the eluted RNA directly in RT-PCR
Use 3
...
E OR store the eluted RNA for later analysis at –80°C
...
Troubleshooting the High Pure protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
For factors that may affect the High Pure
Viral RNA Kit, see page 120
...
Each preparation was used as a template in RT-PCR
...
Figure 26: RT-PCR analysis of MS2
RNA isolated with the High Pure
Viral RNA Kit
...
3
...
The indicated
numbers of molecules per PCR
correspond to assumed quantitative
recovery
...
5 x 107 molecules/PCR;
Lane 2: 3
...
5 x 103 molecules/PCR
Lane 4: 3
...
Nucleic Acid Isolation and Purification Manual
High Pure Viral RNA Kit
Typical result with the kit
References
Akiyama M
...
(2009) J
...
Microbiol
...
et al
...
Gen
...
, 89, 2518 – 2530
Dortmans J
...
F
...
et al
...
Virol
...
et al
...
Virol
...
L
...
(2008) RNA, 14, 2394 – 2406
Raboni S
...
et al
...
Gen
...
, 90, 2166 – 2171
Sato S
...
(2008) J
...
, 82, 9739 – 9752
da Silva D
...
(2010) J
...
Chemother
...
et al
...
Clin
...
, 47, 1906 – 1910
Zhou F
...
(2009) J
...
Microbiol
...
No
...
Then, in the presence of a chaotropic salt (guanidine HCl), viral NA binds selectively to
glass fiber fleece in a special centrifuge tube
...
Finally, low salt
elution removes the NA from the glass fiber fleece
...
Starting material
Research samples may be 200 – 600 µl of:
Serum
Plasma
Whole blood
ell culture supernatant
C
Application
Preparation of highly purified viral nucleic acids (RNA or DNA), which may be used
directly as templates for standard or long-template PCR or RT-PCR
Nucleic acid preparations obtained are suitable for PCR or RT-PCR; they are not
tested for other applications
...
20 min
Hands-on time: <10 min
Results
Yield: 50 µl eluate is enough for 8 – 14 RT-PCRs or 2 – 5 PCRs
...
Benefits
Saves time, because the kit can prepare multiple PCR/RT-PCR templates in just
minutes
...
Minimizes loss of nucleic acids, because the kit removes contaminants without
precipitation, solvent extraction, or other handling steps that can lead to lost or
degraded nucleic acids
...
100
Nucleic Acid Isolation and Purification Manual
High Pure Viral Nucleic Acid Kit
How to use the kit
How to use the kit
I
...
speed
(13,000 x g) for 10 seconds
Discard flowthrough and
Collection Tube
Add 50 µl Elution Buffer
Centrifuge at 8000 x g
for 1 min
Purified Viral Nucleic
Acids
High Pure Kits and Silica Adsorption
101
High Pure Viral Nucleic Acid Kit
How to use the kit
II
...
Therefore, dissolve the Poly(A) Carrier RNA in Elution
Buffer:
E
Add 0
...
Stopper and invert the vial
until all the Carrier RNA (including any that might stick to the rubber stopper) is
completely dissolved
...
Before
each experiment, thaw one tube of reconstituted Carrier RNA and add the
contents to 2
...
This working solution may be stored at +15 to
+25°C, but must be used within a few days
...
Inhibitor Removal Buffer (33 ml)
Note: Add 20 ml absolute ethanol to buffer before use
...
Elution Buffer (30 ml)
High Pure Filter Tubes (100 tubes)
Collection Tubes, 2 ml (400 tubes)
III
...
5 ml, nuclease-free
IV
...
To a nuclease-free 1
...
E Add 200 µl working solution [Carrier RNA-supplemented Binding Buffer]
...
E Incubate for 10 min at +72°C
...
EEE
102
Nucleic Acid Isolation and Purification Manual
High Pure Viral Nucleic Acid Kit
How to use the kit
To transfer the sample to a High Pure Tube:
E Insert one High Pure Filter Tube into one Collection Tube
...
Insert the entire High Pure Tube assembly into a standard tabletop microcentrifuge,
then centrifuge the tube assembly for 1 min at 8000 x g
...
2
E Insert the Filter Tube into a new Collection Tube
...
Centrifuge for 1 min at 8000 x g (as in Step 4)
...
Reinsert Filter Tube into a new
C
ollection Tube
...
E Repeat the centrifugation (as in Step 4)
...
E Repeat the wash step (Step 6)
...
13,000 x g) to remove residual Wash Buffer
...
5 ml
microcentrifuge tube
...
If the starting sample (Step 1) was whole blood, prewarm the Elution Buffer
to +70°C before adding it to the Filter Tube
...
The microcentrifuge tube now contains the eluted, stable viral nucleic acids
...
5 – 6 µl of the eluate for the reverse
transcriptase reaction
...
E OR store the eluted nucleic acids for later analysis at either +2 to +8°C (viral
DNA) or –80°C (viral RNA)
...
Troubleshooting the High Pure protocols
The same troubleshooting procedure can be applied to all High Pure kits
...
For factors that may affect the High Pure
Viral Nucleic Acid Kit, see page 121
...
Each preparation was used as a template in PCR (DNA) or RTPCR (RNA)
...
2
Figure 27: Sensitivity of viral RNA detection
...
20 µl of 100 µl eluates were analyzed by one step
RT-PCR using either Tth DNA Polymerase (in case of HCV and HIV) or a mixture of AMV Reverse Transcriptase
and Taq DNA Polymerase (in case of HGV)
...
Result: Assuming quantitative recovery, detection sensitivity is in the range of 20 – 40 viral copies per PCR
...
G
...
et al
...
Virol
...
et al
...
Chem
...
et al
...
Virol
...
et al
...
Virol
...
et al
...
Med
...
, 58, 878 – 883
He J
...
(2009) J
...
Microbiol
...
et al
...
T
...
(2009) J
...
Microbiol
...
L
...
(2008) J
...
, 82, 11023 – 11044
Wolf S
...
(2010) Appl
...
Microbiol
...
No
...
Viral lysis is accomplished by incubating the sample in a special
Lysis/Binding Buffer in the presence of Proteinase K
...
During the spin, the lysis mixture passes through the glass-fiber fleece of the High Pure
Spin Column, and nucleic acids are bound to the silica fibers
...
During this process, nucleic
acids are purified from salts, proteins, and other impurities by washing and centrifugation steps
...
2
Figure 28: High Pure Large Volume Assembly
...
5 ml without complicated sample pre-processing
...
Starting material
Purification of viral nucleic acids from up to 2
...
Application
The High Pure Viral Nucleic Acid Large Volume Kit is designed to efficiently purify viral
nucleic acids from serum, plasma, or whole blood
...
5 ml
...
For optimal results,
the first step of the isolation process should be performed in a tabletop centrifuge with a
swinging bucket rotor capable of holding 50 ml tubes
...
Time required
Total time: approx
...
Purity: Purified NA is free of intact virus, nucleases, and small cellular components
that interfere with RT-PCR or PCR
...
Use sample volumes up to 2
...
Obtain high-purity nucleic acids
...
ncrease convenience and improve time to result
...
High Pure Kits and Silica Adsorption
105
High Pure Viral Nucleic Acid Large Volume Kit
How to use the kit
How to use the High Pure Extender Assembly
The High Pure Extender Assembly is delivered in single zip-bags
...
Each High Pure Extender is assembled in a 50 ml polypropylene Tube
...
2
Cap of the 50 ml polypropylene tube
High Pure Extender
Locking clip (side a)
Locking clip (side b)
High Pure Filter Tube
50 ml polypropylene tube
Figure 29: High Pure Extender Assembly
Remove the High Pure Extender Assembly from the zip-bags prior to use
...
After sample loading, close the High Pure Extender
Assembly with the 50 ml polypropylene tube cap
...
Nucleic acids are bound to the silica fleece at the
bottom of the High Pure filter tube
...
Discard the tube containing the flow-through
...
Disassembly of the High Pure Extender Assembly
Figure 30: Unscrew the 50 ml polypropylene tube
and remove the High Pure Extender Assembly from
the 50 ml polypropylene tube
...
Nucleic Acid Isolation and Purification Manual
High Pure Viral Nucleic Acid Large Volume Kit
How to use the kit
Figure 32: Secure the High Pure Extender Assembly
with one hand while grasping the locking clip (side a)
of the High Pure Filter Tube cap on the opposite side
with the other hand
...
Figure 33: Rotate the High Pure Extender Assembly
...
2
Figure 34: Remove the High Pure Extender from the
High Pure Filter Tube by tilting the High Pure Extender
away from the High Pure Filter tube toward the side
without the cap
...
However, if you decide to use a centrifuge with fixed-angle rotors, do not centrifuge
High Pure Extender Assembly above 3000 g
...
The remaining sample solution
therefore stays within the High Pure Spin Column Filter Tube
...
For this additional spin remove the High
Pure Spin Column from the High Pure Extender Assembly according to Figures 30
to 35
...
Flow diagram
1 ml serum, plasma or
whole blood
Add 1 ml Binding Buffer
supplemented with Poly (A)
and 250 µl Proteinase K
Mix immediately and
incubate for 15 min at +70°C
...
Remove the High Pure
Filter Tube from the
Extender Assembly
...
Place the Filter
Tube in a new Collection
Tube
Discharge the flowthrough
and Collection Tube
Add 500 µl Inhibitor
Removal Buffer
Centrifuge for 1 min
at 8,000 × g
Add 450 µl Washing Buffer
Centrifuge for 1 min
at 8,000 × g
Add 450 µl Washing Buffer
Discharge the flowthrough
Centrifuge for 1 min
at 8,000 × g
Discharge the flowthrough
Centrifuge for 30 sec max
...
Kit contents
All solutions are clear
...
Warm the
solutions at +15 to +25°C or in a +37°C water bath until the precipitates have
dissolved
...
4 (+25°C)
Poly(A) Lyophilizate, 2 mg poly(A) carrier RNA for binding of RNA
Dissolve poly(A) carrier RNA in 0
...
Prepare 15 µl aliquots
...
5 ml to
2
...
Always prepare freshly before use! Do not store!
2
Proteinase K lyophilizate, 2 × 100 mg for the digestion of proteins
Dissolve Proteinase K in 5
...
Prepare 130 µl
aliquots
Store aliquots at –15 to –25°C, Stable for 12 months
Inhibitor Removal Buffer, 33 ml, 5 M guanidine-HCl, 20 mM Tris-HCl, pH 6
...
5 (+25°C), final concentrations after addition of ethanol
Add 40 ml ethanol p
...
Additional material needed
Absolute ethanol
Standard tabletop centrifuge with swing-bucket rotor capable of 5000 × g centrifugal
force for 50 ml polypropylene tubes
Standard tabletop microcentrifuge capable of 13,000 × g centrifugal force
(e
...
, Eppendorf 5415C or equivalent)
Microcentrifuge tubes, 1
...
Protocol for preparing nucleic acids from 1 ml samples of
serum, plasma, or whole blood (research samples)
1
To a nuclease-free 15 ml falcon tube
E Add 1 ml serum, plasma or whole blood
E
Add 1 ml working solution, freshly prepared, (carrier RNA-supplemented Binding
Buffer)
2
E Add 250 µl Proteinase K solution, and mix immediately
...
2
Add 400 µl Binding Buffer and mix
...
3
Pipette entire sample into the upper reservoir of the High Pure Extender Assembly
...
E Then centrifuge for 5 min at 4,000 × g
...
E Combine the Filter Tube with a new Collection Tube
...
E Add 500 µl Inhibitor Removal Buffer to the upper reservoir of the Filter Tube
...
7
After centrifugation:
E Remove the Filter Tube from the Collection Tube
...
E Discard the Collection Tube including the flowthrough liquid
...
E Centrifuge for 1 min at 8,000 × g and discard the flowthrough
...
E Combine the Filter Tube with a new Collection Tube
...
E Centrifuge for 1 min at 8,000 × g and discard the flowthrough
...
13,000 × g) to remove any residual Wash Buffer
...
Discard the Collection Tube and insert the Filter Tube into a nuclease-free, sterile
1
...
To elute the viral nucleic acids:
E Add 50 µl Elution Buffer to the upper reservoir of the Filter Tube
...
E Centrifuge the tube assembly for 1 min at 8,000 × g
...
You may:
E Either use the eluted nucleic acids directly in PCR (10 — 20 µl DNA eluate) or
RT-PCR (3
...
The High Pure Extender Assembly is designed for sample volumes up to
2
...
The sample buffer compositions for different sample volumes are
listed in the table below
...
5 ml
1 ml
2
2
...
5 ml
1 ml
2
...
2 ml
0
...
V
...
For details on
how to troubleshoot the above protocol, see the General Troubleshooting Procedure for
all High Pure kits on page 113 of this manual
...
High Pure Kits and Silica Adsorption
111
High Pure Viral Nucleic Acid Large Volume Kit
Typical result with the kit
Typical results with the kit
Experiment 1
Figure 35: Generate high-quality
template with excellent linearity in
qRT-PCR
...
Nucleic acids were isolated from
1 ml of spiked plasma using the High
Pure Viral Nucleic Acid Isolation Large
Volume Kit as described in the kit
protocol
...
2
Result: The template purified using the
High Pure Viral Nucleic Acid Isolation
Large Volume Kit generated a highly
specific PCR product that demonstrates
excellent performance and linearity in
qRT-PCR
...
Various amounts (200 µl, 1 ml, 25 ml,
and 5 ml) of serum research samples were spiked with a dilution series (1 x 106 to 1 x 104 copies/ml) of hepatitis
A virus (HAV) particles
...
Five microliters of each sample eluate was analyzed by
LightCycler® System qRT-PCR
...
The experiment also demonstrates that the High Pure
Viral Nucleic Acid Large Volume Kit can accommodate serum sample volumes as large as 5 ml, yielding highly
pure, concentrated nucleic acids
...
112
Nucleic Acid Isolation and Purification Manual
Troubleshooting Procedure for the High Pure Kits
Troubleshooting Procedures
I
...
Then, the cause
may be
...
Low nucleic acid
yield or purity
Kit stored in less
than optimal
conditions
E Store all High Pure kits at +15 to +25°C as soon as they arrive
...
reagents exposed to
E the temperature in your laboratory is lowered overnight,
If
conditions that
warm all buffers at +37°C for 10 to 15 min
...
2
E
Close all reagent bottles tightly after each use to preserve pH,
stability, and freedom from contamination
...
Ethanol not added
to Wash Buffer
E
Add absolute ethanol to all Wash Buffers before using (as
indicated in kit package insert)
...
E
Always mark the Wash Buffer vial to indicate whether ethanol
has been added or not
...
Water has the wrong E you use PCR grade water to elute nucleic acids from Filter
If
pH
Tube, be sure the water has the same pH as the Elution Buffer
supplied in the kit
...
Kit
...
Recovery can be improved by incubating the column filter
with elution buffer for 1 to 5 min after addition of elution
buffer
...
Remove High Pure Filter Tube from tube containing eluted
restriction enzyme coeluted with the
sample and spin sample tube for 1 min at maximum speed
...
Transfer supernatant to a new tube without disturbing the
enzymes
glass fibers at the bottom of the original tube
...
Factors that may affect all High Pure kits, continued
If you get
...
And you should
...
nucleic acid, scatter
light
Samples “pop” out
of wells in agarose
gel
Eluate contains
ethanol (from the
Wash Buffer)
E
...
E mpty Collection Tube, reinsert Filter Tube in emptied
2
...
K
II
...
Then, the cause
may be
...
To solubilize the lyophilized Proteinase K completely:
E 1
...
5 ml of PCR grade water into the glass vial containP
ing lyophilized Proteinase K
...
topper and invert the vial until all the lyophilizate (includS
ing any stuck to the rubber stopper) is dissolved
...
liquot the reconstituted enzyme, mark each aliquot with
A
the date of reconstitution, and store at –15 to –25°C
...
E fter adding Proteinase K to sample, mix immediately
...
Low yield
from tissue
Incomplete
P
roteinase K
digestion
E Cut tissue into small pieces before digestion and lysis
...
E
Incubate with Proteinase K for 3 – 4 h, then add a fresh
aliquot of Proteinase K (30 µl) and incubate another 1 – 2 h
...
Cells not lysed
E
Make sure your cells can be lysed by lysozyme or lyticase
...
or lyticase (yeast)
E alternative lysis procedures, such as mechanical grinding,
Use
vortexing with glass beads, boiling, or repeated freeze-thaw
...
Factors that may affect the High Pure PCR Template
P
reparation Kit, continued
If you get
...
And you should
...
2
E only small pieces of tissue (20 – 40 mg) in the procedure,
Use
or homogenize the tissue sample
...
E
Repeat purification protocol by mixing 200 µl eluate with
200 µl Binding Buffer, then 100 µl isopropanol
...
Omit Proteinase K digestion and +72°C incubation
...
Factors that may affect the High Pure PCR Cleanup Micro
Kit
If you get
...
And you should
...
Do
Alkaline pH is required for optimal elution
...
Use
Elute DNA with two volumes of Elution Buffer (>10 µl each)
...
Concentration of
Low concentrations Verify PCR result by agarose gel electrophoresis before starting
DNA in the eluate is of amplified DNA purification procedure
...
material
Short read length
in sequencing
reactions
Copurification of
DNA binding
proteins
Use the Expand High Fidelity PCR System for template synthesis
...
primer and primerdimer
K
High Pure Kits and Silica Adsorption
115
Troubleshooting Procedure for the High Pure Kits
IV
...
Then, the cause
may be
...
conditions that
lessened their
effectiveness
2
And you should
...
mixed
Too little Binding
Buffer used
E Make sure the ratio of PCR product to Binding Buffer is 1:5
...
Concentration
of DNA in
eluate too low
Too much Elution
Buffer
E Decrease volume of Elution Buffer
...
il overlay, wax, and gel loading dye do not interfere with
O
the purification procedure
...
Do
K
V
...
Then, the cause
may be
...
Low plasmid
yield
Too few cells in
starting material
E Grow E
...
0 – 1
...
Incomplete cell lysis E sure the E
...
E
Make sure the lysate is clear and viscous after the lysis step
(incubation with Lysis Buffer)
...
The precipitate should pellet completely during centrifugation
...
Plasmid is degraded High levels of
or no plasmid
n
uclease activity
obtained
o not centrifuge the Filter Tube after this step
...
E optional Wash Buffer I (Step 7 of protocol) to eliminate
Use
nuclease activity in E
...
EEE
116
Nucleic Acid Isolation and Purification Manual
Troubleshooting Procedure for the High Pure Kits
V
...
Then, the cause
may be
...
RNA present in
final product
RNase not completely dissolved
To solubilize the lyophilized RNase completely:
E 1
...
E 2
...
E 3
...
E 4
...
Too many cells in
starting material
2
Reconstituted mixture is stable for 6 months when stored
properly
...
coli culture as
Do
starting material
...
step
not vortex!
Do
Additional
band running
slightly faster
than supercoiled
plasmid is seen
on gels
Denatured plasmid E
Reduce the incubation time during Step 3 (lysis step) of the
in final product
protocol
...
Factors that may affect the High Pure RNA Isolation Kit
If you get
...
And you should
...
Be
E
Process starting material immediately or store it at –80°C until
it can be processed
...
Cultured cells
not completely
resuspended
E
Resuspend cell pellet completely in PBS before starting
procedure
...
Factors that may affect the High Pure RNA Isolation Kit,
continued
If you get
...
And you should
...
ipette 0
...
2
...
3
...
Reconstituted DNase is stable for 12 months when stored
properly
...
DNase not evenly
distributed in
High Pure Filter
Tube
E
When pipetting, distribute the working DNase solution evenly
over the glass fleece in the Filter Tube
...
5 ml whole blood,
Do
108 yeast, or 109 bacteria
...
K
VII
...
Then, the cause
may be
...
Frozen tissue should not be allowed
optimal conditions
to thaw during handling prior to disruption in Lysis/Binding
Buffer
...
E
Addition of 0
...
High levels of RNase E careful to create an RNase-free working environment
...
E eluate RNA directly in downstream procedures or store it
Use
immediately at –80°C
...
E
Reduce amount of starting material and/or increase the
amount of Lysis/Binding Buffer
...
Factors that may affect the High Pure RNA Tissue Kit,
continued
If you get
...
And you should
...
g
...
Too much
starting material
E
Reduce amount of starting material and/or increase the
amount of Lysis/Binding Buffer
...
K
VIII
...
Then, the cause
may be
...
Low RNA yield or
purity
High levels of RNase E careful to create an RNase-free working environment
...
E eluted RNA directly in downstream procedures or store it
Use
immediately at –80°C
...
difficult to pipette, zation
low RNA yield
Low yield and /or
Nucleic acid is
bad performance in cross-linked to
RT-PCR
impurities
Too much starting
material
Clogged filter tube
E
Increase Proteinase K digestion time in step 1 of the RNA
Isolation Protocol to 16 h (overnight)
...
Insufficient disrup- E
E
...
, increase the disruption time for the rotor stator homogtion and/or homogenizer or pass through syringe/needle several times
...
K
High Pure Kits and Silica Adsorption
119
Troubleshooting Procedure for the High Pure Kits
IX
...
Then, the cause
may be
...
Low RNA yield and Inappropriate
purity
storage and handling of tissue
2
E fresh tissue and disrupt immediately or flash-frozen tissue
Use
stored at –70°C
...
g
...
Too much
starting material
Clogged filter tube
E
Reduce amount of starting material and/or increase the
amount of Lysis/Binding Buffer
E
Reduce amount of starting material and/or increase the
amount of Lysis/Binding Buffer
K
X
...
Then, the cause
may be
...
Carrier RNA
not completely
dissolved
And you should
...
4 ml Elution Buffer into the glass vial containing
1
...
E topper and invert the vial until all the lyophilizate (includ2
...
E liquot the carrier RNA mark each aliquot with the date of
3
...
haw one vial with 4 µl carrier RNA and mix thoroughly
T
with 5 ml Binding Buffer
...
Factors that may affect the High Pure Viral Nucleic Acid Kit
or the High Pure Viral Nucleic Acid Kit Large Volume
If you get
...
And you should
...
Low DNA or RNA
yield
Carrier RNA
not completely
dissolved
E solubilize the lyophilized Poly(A) Carrier RNA completely:
To
1
...
5 ml Elution Buffer into the glass vial containing
lyophilized Carrier RNA
...
Stopper and invert the vial until all the lyophilizate (includ
ing any stuck to the rubber stopper) is dissolved
...
liquot the Carrier RNA, mark each aliquot with the date of
A
reconstitution, and store at –15 to –25°C
...
5 ml Binding Buffer
...
ing lyophilized Proteinase K
...
ing any stuck to the rubber stopper) is dissolved
...
A
the date of reconstitution, and store at –15 to –25°C
...
K
High Pure Kits and Silica Adsorption
121
Agarose Gel DNA Extraction Kit
Agarose Gel DNA Extraction Kit
for recovery of up to 100 DNA fragments from agarose gel slices
Cat
...
11 696 505 001
Principle
2
A solubilization buffer that contains a chaotropic salt (sodium perchlorate) dissolves an
agarose gel slice that contains a DNA fragment
...
The DNA remains bound while a series
of rapid “wash-and-spin” steps remove contaminating small molecules
...
The process does not require DNA
precipitation, organic solvent extractions, or extensive handling of the DNA
...
4 – 100 kb) from PCR or other enzymatic
reactions
Single-stranded oligonucleotides (>20 nucleotides)
Kit contains enough reagents for purification of 100 DNA fragments from gel slices
containing up to 2% agarose
...
Application
Preparation of concentrated, purified DNA, which may be used directly for labeling,
sequencing, cloning, PCR, and other procedures requiring concentrated DNA
Time required
Total time: 60 min (+ gel running time)
Hands-on time: <30 min
Results
Recovery:
DNA fragments (0
...
5 kb long), approx
...
Large DNA fragments (10 – 100 kb), approx
...
Single-stranded oligonucleotides (>20 bases), approx
...
Purity: Purified DNA is free of agarose and all inhibitors of downstream procedures
(small molecules, nucleotides, proteins)
...
Minimizes DNA loss, because the kit recovers DNA from agarose without solvent
extraction, precipitation, or other steps that can lead to lost or degraded DNA
...
Increases lab safety, because the kit does not use hazardous organic solvents
...
Flow diagram
Separate DNA electrophoretically in an agarose gel
Cut gel slice containing DNA
Solubilize gel slice and bind DNA to Silica Matrix
2
Pellet Silica Matrix and discard the supernatant
Wash Silica Matrix (3 x), pelleting after each wash
Drain residual liquid from Silica Matrix
Elute DNA from the Silica Matrix
II
...
Additional materials needed
Agarose LE, MP or MS (Roche Applied Science)
Gel running buffer:
TAE Buffer (40 mM Tris-acetate, 1 mM EDTA), pH 8
...
5
TBE Buffer (45 mM Tris-borate, 1 mM EDTA), pH 8
...
5
Electrophoresis equipment
Sterile scalpel
High Pure Kits and Silica Adsorption
123
Agarose Gel DNA Extraction Kit
How to use the kit
Absolute ethanol
Standard tabletop microcentrifuge
Microcentrifuge tubes, sterile, 1
...
0 ml
TE Buffer (10 mM Tris-HCl, 0
...
0 – 8
...
Protocol for preparing DNA from agarose gels
Gels containing >2% agarose may be solubilized with twice the standard amount of
Solubilization Buffer
...
Separate the DNA of interest (for example, PCR product) electrophoretically:
E Prepare an agarose gel with Roche Applied Science agarose
...
E Use 1 x TAE or 1 x TBE as running buffer
...
After gel electrophoresis:
E Stain the gel with ethidium bromide
...
Cut the DNA band from the gel with a sharp scalpel or razor blade
...
Place the agarose gel slice into a sterile microcentrifuge tube (1
...
0 ml) of known
weight, then reweigh the tube
...
Resuspend the Silica Matrix to produce a homogeneous suspension
...
5 µg DNA, add 10 µl of the resuspended Silica Matrix
to the agarose gel slice
...
5 µg DNA, add 4 µl of the resuspended Silica Matrix to
the agarose gel slice for every µg of DNA in the gel
...
E Incubate the suspension for 10 min at +56 to +60°C
...
Pellet the Silica Matrix (which contains the DNA) by centrifuging the tube in a
tabletop microcentrifuge at maximum speed for 30 s
...
Resuspend the Silica Matrix by adding 500 µl of Nucleic Acid Binding Buffer and
vortexing
...
Wash the Silica Matrix by adding 500 µl of Wash Buffer and vortexing
...
Repeat the wash steps (Steps 11 and 12)
...
Do not dry the pellet under vacuum, since overdrying will reduce the amount of
DNA recovered from the matrix
...
0
– 8
...
E Resuspend the pellet by vortexing
...
5 kb), incubate at +56° to +60°C for
15 – 20 min
...
After the elution is complete:
E Pellet the Silica Matrix by centrifuging the tube in a microcentrifuge at maximum
speed for 30 s
...
Avoid transferring any Silica Matrix with the supernatant
...
Troubleshooting the Agarose Extraction protocol
If you get
...
And you should
...
Ethanol from the
Wash Buffer
remains during
DNA elution
E
Before eluting the DNA, completely remove Wash Buffer with a
Pasteur pipette, then air dry the pellet thoroughly
...
0 – 8
...
Large DNA fragments adsorbed
strongly to Silica
Matrix
E Perform elution (Step 16) at +56 to +60°C for 15 – 20 min
...
Vortex the tube every 2 – 3 min during incubation
...
Troubleshooting the Agarose Extraction protocol, continued
If you get
...
And you should
...
Silica Matrix not
E
Recentrifuge DNA solution and transfer supernatant to a new
completely removed
microcentrifuge tube
...
Binding Buffer not
completely removed
and chaotropic salts
inhibit reaction
...
DNA fragments (0
...
Isolated fragments were displayed on a new agarose gel
...
5 kb fragment
Lane 4: 6
...
3 kb fragment
Lane 6: 2
...
0 kb fragment
Lane 8: 0
...
References
Chew, J
...
et al
...
Cell
...
, 25, 6031 – 6046
Dobi, A
...
(2006) J
...
, 281, 28889 – 28900
Lannoo, N
...
(2006) J
...
Bot
...
et al
...
Gen
...
, 86, 719 – 725
Platara, M
...
(2006) J
...
Chem
...
A
...
(2007) Am
...
Pathol
...
T
...
(2006) Clinical and Applied Thrombosis/Hemostasis, 12, 77 – 84
Zhao J
...
et al
...
Envir
...
, 74, 1980 – 1987
126
Nucleic Acid Isolation and Purification Manual
High Pure 96 UF Cleanup Kit
High Pure 96 UF Cleanup Kit
for high-throughput purification of PCR products by ultrafiltration
Cat
...
04 422 694 001 (2 x 96 purifications)
Principle
The kit uses ultrafiltration and size exclusion to separate small from large molecules
...
) that may interfere with
downstream applications readily pass through a membrane (and into a waste container),
while PCR products remain atop the membrane
...
Starting material
The kit can recover 20 - 300 μl PCR products that range from 100 bp to >10 kb
...
Time required
Manual processing on vacuum manifolds or microplate centrifuges takes approx
...
Automated processing depends on the liquid handling instrument used; it can be as little as 15 min
...
8 ± 0
...
Typically, 90% of a 1 kb fragment can be recovered in 25 µl elution buffer
...
Benefits
Flexible; works well with vacuum manifolds, microplate centrifuges, and common
liquid handling instruments
...
Suitable for high volume laboratories; purifies up to 96 samples in less than 20
minutes
...
Convenient; samples are applied and removed from the top
...
High Pure Kits and Silica Adsorption
127
High Pure 96 UF Cleanup Kit
How to use the kit
How to use the kit
I
...
Kit content
2 High Pure 96 UF Cleanup Plates
100 ml Wash Buffer
2 Waste Plates
100 ml Resuspension Buffer
2 Elution Plates with self-adhering foil
128
Nucleic Acid Isolation and Purification Manual
High Pure 96 UF Cleanup Kit
How to use the kit
III
...
g
...
Isolation Protocol
IVa
...
You will have to increase
the filtration time to process larger volumes (up to 300 µl)
...
1
Pipette 20 to 100 µl PCR reaction mix directly onto the membrane in one well of the
High Pure 96 Cleanup Plate
...
Unused wells of the
plate may be left open
...
2
Remove contaminants by ultrafiltration
...
Indicated times are approximate
...
Make sure that all liquid has passed through the
membrane (membrane will appear dry)
...
400 to
600 mbar for 10 to 15 min
...
Dispense 100 µl Wash
Buffer into each well
...
Apply vacuum for an
additional 30–60 s
...
However, if you have
problems with downstream applications or want to ensure removal of small
molecules, we recommend using the extra wash step
...
Add 25 to 100 µl Resuspension Buffer
directly to the center of the membrane in each well
...
(We recommend using 50 μl Resuspension Buffer for resuspension on a shaker)
...
7
Use purified product directly in downstream applications or seal Elution Plate with foil
for storage of the resuspended DNA samples
...
Isolation Protocol Using a Microtiter Plate Centrifuge
1
Pipette 20 to 100 µl PCR reaction mix directly onto the membrane in one well of the
High Pure 96 UF Cleanup Plate
...
Unused wells of the
plate may be left open
...
2
Place the plate onto the Waste Collection Plate (supplied with the kit)
...
Indicated times are approximate
...
Make sure that all liquid has passed through the
membrane (membrane will appear dry)
...
Centrifuge the assembly at 4,500 x g for 5–10 min at +15 to +25°C
...
After the first spin, minute amounts of liquid
(containing salts etc
...
4
Add 25 to 100 µl Resuspension Buffer directly to the center of the membrane in each
well
...
5
Incubate the Resuspension Buffer on the membrane for 5–10 min OR
Resuspend DNA by pipetting the liquid up and down 5–10 times mixing OR
Resuspend DNA by placing the Cleanup Plate on a microplate shaker for 2 to 5 min
...
6
Use a pipette to remove the resuspended, purified product from the Cleanup Plate
and transfer it to the clean Elution Plate
...
130
Nucleic Acid Isolation and Purification Manual
High Pure 96 UF Cleanup Kit
How to use the kit
V
...
Then, the cause
may be
...
Low DNA recovery
Elution conditions
not optimal
E
Increase number of mixing steps
...
Did not use enough E
Recommended amount of Resuspension Buffer:
Resuspension Buffer at least 25 µl for manual purification or
least 50 µl for automated purification or resuspension on a
at
shaker
DNA fragments
E
Dispense Resuspension Buffer or PCR grade water onto
dried on membrane
membrane and incubate 15 to 30 min at +15 to +25°C to allow
DNA to rehydrate before trying to recover it
...
2
E
Pipette the resuspended DNA up and down at least 5-10 times
...
the inner wall of
Make sure that no sample material sticks to the side of the
well
wells
...
E
Perform optional washing step
Slow filtration
PCR buffer contains E
Although detergents are effectively removed by ultrafiltration
detergents
on the High Pure 96 UF Cleanup Plate, the presence of
detergent in commercially available PCR buffers may lead to
increased filtration time
...
Use
K
High Pure Kits and Silica Adsorption
131
High Pure 96 UF Cleanup Kit
Typical results with the kit
Typical results with the kit
2
Figure 38: Purification of small and large PCR products with the High Pure 96 UF Cleanup Kit and
competing products
...
Lanes 1: 1
...
2 kb PCR fragment, lanes 3: 600 bp PCR fragment,
lanes 4: 350 bp PCR fragment, lanes 5: 165 bp PCR fragment
...
)
Figure 39: Comparison of purification via vacuum (lanes 7-12) and centrifugation (lanes 1-6, 13-18)
...
Samples analyzed in lanes 1-6 and 13-18 were purified on different High Pure 96
UF Cleanup Plates
...
(M: DNA Molecular Weight Marker XIV
...
L
...
(2010) Infect
...
, 78, 1314 − 1325
Victor M and Walter T
...
et al
...
, 19, 667 − 673
132
Nucleic Acid Isolation and Purification Manual
Ion Exchange
Chromatography
Overview of Ion Exchange Chromatography
Genopure Plasmid Midi Kit
Genopure Plasmid Maxi Kit
Genopure Buffer Set for Low-Copy Number Plasmids
134
136
143
150
3
Overview of Ion Exchange Chromatography
Overview of Ion Exchange
C
hromatography
This chapter describes our kits for midi and maxi preparation of ultrapure plasmid
DNA
...
Avoids use of phenol/chloroform extraction
3
Uses gravity flow columns, no centrifugation required
For a general overview of these products, continue reading this article, or for detailed
information of the single product turn to the page which describes the product in detail
...
150
The isolation method is based on a modified alkaline lysis protocol and can be divided
into the following steps:
Harvest and disruption of the bacterial cells
Precipitation of the bacterial ”chromosomal” DNA
Clarification of the bacterial lysate
Adsorption of the plasmid DNA to the column matrix
Removal of residual impurities by wash steps
Elution of the plasmid DNA by high salt conditions
Concentration and salt removal by alcohol precipitation
The isolation method is optimized for cultures grown in LB media; other rich media
may require increased volumes of Suspension-, Lysis or Neutralization Buffer, and an
additional wash step
...
The yield of plasmid DNA preparations is dependent on several parameters, e
...
, quality
of the bacterial culture growth, amount of used culture suspension for the preparation,
plasmid type used etc
...
The typical yield of low copy number plasmids is about 0
...
It is recommended to use the supplementary Genopure Buffer
Set for low copy number plasmids in combination with the respective Genopure Plasmid
Kit
...
In approximately 2 min (midi) or 10 min (maxi), respectively,
of unattended running cellular debris and potassium dodecyl sulfate precipitates are
held back by the filter thereby avoiding shearing of large DNA constructs
...
Figure 40: Genopure plasmid purification
procedures
...
3
Ion Exchange Chromatography
135
Genopure Plasmid Midi Kit
Genopure Plasmid Midi Kit
for up to 20 preparations of plasmid DNA in medium scale
Cat
...
03 143 414 001
Principle
3
The isolation procedure is based on a modified alkaline lysis protocol and can be divided
into the following steps:
The bacteria are partially lysed, allowing the plasmid DNA to escape the cell wall into the
supernatant
...
coli chromosomal DNA is trapped in the cell wall
...
The bound plasmid DNA is washed to remove contaminating bacterial components
...
This is a commonly used method that generates highly purified plasmid DNA (free of
RNA contamination)
...
coli cultures transformed with a high copy number plasmid
...
coli cultures transformed with a low copy number plasmid
10
Application
(at a density of 2 – 6
...
This kit is used to prepare plasmid DNA in medium quantities known as ”midi preps”
...
The
kit is designed for the isolation of up to 100 µg of plasmid DNA from bacterial culture
...
The quality of the
plasmid DNA is better than plasmid DNA obtained by 2 x CsCl gradient centrifugation
...
g
...
Time required
Total time: 60 min including a filtration step after the alkaline lysis
...
Results
Purity: Plasmid DNA is ready-to-use even in demanding down-stream applications
...
coli strain and density of the cell culture
...
Application: The purified plasmid has been used for PCR, sequencing and trans
fection with excellent results
...
Ready to use because all reagents provided with the kit
...
Parallel processing because of use of high speed gravity flow columns
...
136
Nucleic Acid Isolation and Purification Manual
Genopure Plasmid Midi Kit
How to use the kit
How to use the kit
I
...
coli
culture, at 3000 – 5000 x g
at +2 to +8°C for 10 min
Resuspend pellet in 4 ml
Suspension Buffer/RNase
Add 4 ml Lysis Buffer
Discard supernatant
Mix gently, incubate
at +15 to +25°C
2 – 3 min
Add 4 ml chilled
Neutralization Buffer
Mix gently by inversion,
incubate 5 min on ice
Clear bacterial lysate by
filtration (or by centrifugation), transfer flowthrough
(or supernatant) to preequilibrated column
3
Add 5 ml Wash Buffer
Discard pellet
Allow the column to empty
by gravity flow and repeat
this wash step
Discard flowthrough
Add 5 ml Elution Buffer
Allow the column to empty
by gravity flow and collect
flowthrough
Add 3
...
Kit contents
Suspension Buffer (100 ml) for suspension of bacterial cell pellets
RNase A (12 mg) for dissolution in Suspension Buffer
Lysis Buffer (100 ml) for bacterial cell lysis
Neutralization Buffer (100 ml) to form a stable cellular debris precipitate
Equilibration Buffer (70 ml) for equilibrating the columns prior to use
Wash Buffer (250 ml) for removal of residual impurities
Elution Buffer (125 ml) for plasmid elution
3
NucleoBond AX 100 Columns (20 columns) for the isolation step
Folded filters (20 filters) to eliminate a centrifugation step and to remove cellular
debris
Sealing rings (10 rings) to station the columns in test tubes
III
...
Protocol for preparing high copy number plasmid DNA
Step Action
Time / x g /
Temperature
E
Centrifuge bacterial cells from 5 – 30 ml E
...
E
Discard the supernatant
...
E
Add 4 ml Lysis Buffer to the suspension and mix gently by inverting the
tube 6 to 8 times and incubate
...
Do not incubate for more than 5 min to prevent the release of
chromosomal DNA from the cell debris
...
E
Immediately mix the suspension gently by inverting the tube 6 to 8 times
until a homogeneous suspension is formed
...
5 min on ice
The solution becomes cloudy and flocculent precipitate will form
...
V E
Centrifuge at high speed
E
Directly after centrifugation carefully remove the supernatant from the
white precipitate and proceed with step 5
...
>30 min / >12,000 x g /
+2 to +8°C
E
Moisten the filter with a few drops of Equilibration Buffer or PCR grade
water
...
The SDS is removed with the Neutralization Buffer (white precipitate) and should not be loaded onto the column
...
E
Mount the sealing ring on the column as shown in Figure 41 to fix the
column in the Collection Tube
...
E Equilibrate the column with 2
...
E Allow the column to empty by gravity flow
...
E Load the cleared lysate of step 4 onto the equilibrated column
...
E Discard the flowthrough
...
E Allow the column to empty by gravity flow
...
E Repeat step 7
...
EEE
Ion Exchange Chromatography
139
Genopure Plasmid Midi Kit
How to use the kit
IV
...
E Elute the plasmid with 5 ml Elution Buffer
...
3
E The collected flowthrough contains the plasmid
...
6 ml isopropanol equilibrated to
+15 to +25°C
...
30 min / 15,000 x g /
+2 to +8°C
E Carefully discard the supernatant
...
+2 to +8°C
E Centrifuge at high speed
...
E Air-dry the plasmid DNA pellet
...
K
V
...
Then, the
cause may be
...
Low nucleic acid
yield or purity
Kit stored under
non-optimal
conditions
E
Store kit at +15 to +25°C at all times upon arrival
...
Reagents and
samples not
completely
mixed
E
Always mix the sample tube well after addition of each reagent
...
E
Close all reagent bottles tightly after each use to preserve pH,
stability and freedom from contamination
...
Non-optimal
E Use the Elution Buffer of the kit
...
Salt
is required
for optimal
elution
EEE
140
Nucleic Acid Isolation and Purification Manual
Genopure Plasmid Midi Kit
How to use the kit
V
...
Then, the
cause may be
...
Low plasmid
yield
Too few cells in
starting material
E Grow E
...
Incomplete
cell lysis
E
Ensure the E
...
E
Make sure the lysate is clear and viscous after the lysis step
(incubation with Lysis Buffer)
...
Lysate did not bind E
Pre-equilibrate the column by adding Equilibration Buffer
completely
before adding sample
...
Pipette 1 ml of Suspension Buffer into the glass vial contain
ing lyophilized RNase
...
Stopper and invert the vial until all the lyophilizate (includ
ing any stuck to the rubber stopper) is dissolved
...
Transfer all the reconstituted RNase back into the Suspen
sion Buffer and mix thoroughly
3
4
...
Reconstituted mixture is stable for 6 months when stored
properly
...
RNase present
in final product
RNase not
completely
dissolved
E See suggestions under ”RNA present in final product” above
...
Denatured
plasmid in final
product
E
Reduce the incubation time during step 2 (lysis step) of the
protocol
...
Plasmid Preparation
Method
Endotoxin
(EU/µg)
Transfection efficiency
(%)
Genopure
4 – 10
100
2-fold CsCl
0
...
3
80
Silica-Matrix/Gel Slurry
> 1000
> 30
Table 3: Mean values from several experiments
...
Equal volumes
of diluted reaction solutions were separated by
SDS-PAGE, then blotted
...
Molecular Weight
Marker [kD]
100
75
45
30
GFP
20
Genopure
Genopure
Plasmid Maxi Plasmid Midi
References
Ausubel, F
...
et al
...
) (1991) Current Protocols in Molecular Biology, Wiley Interscience, New York
Birnboim, H
...
and Doly, J, (1979) Nucl
...
7, 1513 – 1522
Brand O
...
et al
...
Mol
...
, 18, 1704 – 1713
Frias M
...
et al
...
Bacteriol
...
et al
...
Reprod
...
H
...
M
...
(2008) J
...
, 82, 5161 – 5166
Sambrook, J
...
(1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbour Laboratory Press
Song S
...
(2009) Clin
...
, 15, 622 – 631
Takeshi O
...
(2009) J
...
Chem
...
et al
...
Acids Res
...
No
...
The larger E
...
The lysate is
cleared of cellular debris and the plasmid DNA containing fraction is added to the
column
...
The plasmid DNA is eluted and precipitated to remove salt and to concentrate the
eluate
...
Starting material
30 – 150 ml E
...
– 500 ml E
...
0 A600 units per ml bacterial culture)
...
Using a modified alkaline lysis method highly purified plasmid DNA is generated
...
Depending on the copy number of the plasmids use either 30 to 150 ml (high copy
number) or 100 to 500 ml (low copy number) bacterial suspension
...
Therefore the plasmid DNA is suitable for all molecular biology applications e
...
,
transfection, PCR, restriction analysis/Southern blotting, sequencing and cloning
...
Hands-on time: Minimal hands-on time required (about 10 min)
...
Yield: Depending on E
...
Comparable to
traditional purification methods
...
Benefits
Avoids organic or toxic materials as no phenol or chloroform, CsCl and ethidium
bromide needed
...
eliable quality because better than 2 x CsCl
...
P
ll plasmid sizes can be isolated even BAC DNA
...
Flow diagram
Centrifuge 30 – 150 ml
E
...
Kit contents
Suspension Buffer (150 ml) for suspension of bacterial cell pellets
RNase A (15 mg) for dissolution in Suspension Buffer
Lysis Buffer (150 ml) for bacterial cell lysis
Neutralization Buffer (150 ml) to form a stable cellular debris precipitate
Equilibration Buffer (70 ml) for equilibrating the columns prior to use
Wash Buffer (370 ml) for removal of residual impurities
Elution buffer (200 ml) for plasmid elution
NucleoBond AX 500 Columns (10 columns) for the isolation step
Folded filters (10 filters) to eliminate a centrifugation step and to remove cellular
debris
Sealing rings (5 rings) to station the columns in test tubes
III
...
Protocol for high copy number plasmid DNA
Step Action
Time / x g /
Temperature
E
Centrifuge bacterial cells from 30 – 150 ml E
...
5 – 10 min / 3000 – 5000 x g /
+2 to +8°C
E Discard the supernatant
...
E
Add 12 ml Lysis Buffer to the suspension and mix gently by inverting the
tube 6 to 8 times and incubate
...
Do not incubate for more than 5 min to prevent the release of chromosomal DNA from the cell debris
...
E
Immediately mix the suspension gently by inverting the tube 6 to 8 times
until a homogenous suspension is formed
...
5 min on ice
The solution becomes cloudy and flocculent precipitate will form
...
V E
Centrifuge at high speed
E
Directly after centrifugation carefully remove the supernatant from the
white precipitate and proceed with step 5
...
Put
>45 min / >12,000 x g /
+2 to +8°C
E
Moisten the filter with a few drops of Equilibration Buffer or PCR grade
water
...
The SDS is removed with the Neutralization Buffer (white precipitate)
and should not be loaded onto the column
...
E
Mount the sealing ring on the column as shown in Figure 44 to fix the
column in the Collection Tube
...
E
Equilibrate the column with 6 ml Equilibration Buffer
...
E
Discard the flowthrough
...
E
Allow the column to empty by gravity flow
...
E
Wash the column with 16 ml Wash Buffer
...
E
Discard the flowthrough
...
E
Discard flowthrough and Collection Tube
...
Protocol for high copy number plasmid DNA, continued
Step Action
Time / x g /
Temperature
E
Re-insert the column into a new Collection Tube capable of withstanding
high speed centrifugation (15,000 x g)
...
E
Allow the column to empty by gravity flow
...
E
Precipitate the eluted plasmid DNA with 11 ml isopropanol equilibrated to
+15 to +25°C
...
E
Carefully discard the supernatant
...
E
Centrifuge at high speed
...
30 min / 15,000 x g /
+2 to +8°C
3
+2 to +8°C
10 min / >15,000 x g /
+2 to +8°C
E
Air-dry the plasmid DNA pellet
...
K
V
...
Then, the
cause may be
...
Low nucleic acid
yield or purity
Kit stored under
non-optimal
conditions
E
Store kit at +15 to +25°C at all times upon arrival
...
Reagents and
samples not
completely
mixed
E
Always mix the sample tube well after addition of each reagent
...
E
Close all reagent bottles tightly after each use to preserve pH,
stability and freedom from contamination
...
Non-optimal
E the Elution Buffer of the kit
...
Salt
is required
for optimal elution
EEE
Ion Exchange Chromatography
147
Genopure Plasmid Maxi Kit
How to use the kit
V
...
Then, the
cause may be
...
Low plasmid
yield
Too few cells in
starting material
E
Grow E
...
Incomplete
cell lysis
E
Ensure the E
...
E
Ensure the lysate is clear and viscous after the lysis step
(incubation with Lysis Buffer)
...
Lysate did not bind Pre-equilibrate the column by adding Equilibration Buffer before
completely
adding sample
...
ipette 1 ml of Suspension Buffer into the glass vial containP
ing lyophilized RNase
...
topper and invert the vial until all the lyophilizate (includS
ing any stuck to the rubber stopper) is dissolved
...
ransfer the reconstituted RNase back into the Suspension
T
Buffer and mix thoroughly
...
ark the reconstitute mixture (enzyme and buffer) with the
M
date of reconstitution and store at +2 to +8°C
...
Genomic DNA
present in final
product
Genomic DNA
E
Vortexing the preparation after addition of Lysis Buffer should
sheared during lysis
be avoided
...
RNase present in
final product
RNase not completely dissolved
...
See
Too many
cells in starting
material
...
Do
Additional
band running
slightly faster
than supercoiled
plasmid is seen
on gels
Denatured plasmid E
Reduce the incubation time during step 2 (lysis step) of the
in final product
...
K
148
Nucleic Acid Isolation and Purification Manual
Genopure Plasmid Maxi Kit
Typical results with the kit
Typical results with the kit
1
2
3
4
5
6
7
8
Figure 45: 1% 1xTAE gel with pUC clones purified with Genopure Plasmid Kit from different E
...
This gel shows that independent from E
...
Even from strains with higher endonuclease levels no smear, RNA, genomic DNA or
linear plasmid DNA is detectable
...
3
Lanes 1 and 2: JM110
Lanes 3 and 4: Top 10F’ (Invitrogen)
Lanes 5 and 6: BL21
Lanes 7 and 8: DH5a
For further results see page 142
References
Ausubel, F
...
et al
...
) (1991) Current Protocols in Molecular Biology, Wiley Interscience, New York
Birnboim, H
...
and Doly, J, (1979) Nucl
...
7, 1513 – 1522
Brand O
...
et al
...
Mol
...
, 18, 1704 – 1713
Frias M
...
et al
...
Bacteriol
...
et al
...
Reprod
...
H
...
M
...
(2008) J
...
, 82, 5161 – 5166
Sambrook, J
...
(1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbour Laboratory Press
Song S
...
(2009) Clin
...
, 15, 622 – 631
Takeshi O
...
(2009) J
...
Chem
...
et al
...
Acids Res
...
No
...
Starting material
The set provides enough additional buffer for:
Maxi preps with the Genopure Plasmid Maxi Kit (each for isolation of up to
20
500 µg plasmid DNA from 100 to 500 ml bacterial culture), or
Midi preps with the Genopure Plasmid Midi Kit (each for isolation of up to
60
100 µg plasmid DNA from 10 to 100 ml bacterial culture)
...
Doubling the volume of the Suspension, Lysis and Neutralization buffers will help
ensure an efficient yield of low copy number plasmids from bacterial culture
...
Time required
Total time 60 to 75 min (combined use with the Genopure Mini or Maxi Kit) including a
filtration step after alkaline lysis
...
C
Helps the Genopure Plasmid Midi and Maxi Kits enhance plasmid yield
...
E
150
Nucleic Acid Isolation and Purification Manual
Solution-based Isolation
Overview of Solution-based Isolation
DNA Isolation Kit for Mammalian Blood
DNA Isolation Kit for Cells and Tissues
TriPure Isolation Reagent
152
154
162
170
4
Overview of Solution-based Isolation
Overview of Solution-based Isolation
This chapter describes three specialized products
...
All of these products use methods that:
Can be completed in minutes or hours, instead of days
Can process multiple samples simultaneously
Require less handling of potentially hazardous samples
Minimize the use of toxic organic solvents
For a quick overview of each of these products, continue reading this article
...
Traditionally, preparing such DNA from blood required removal
of hemoglobin by labor intensive methods such as density gradients and removal of
protein and lipids with hazardous solvents such as phenol and chloroform (Sambrook et
al
...
The DNA Isolation Kit for Mammalian Blood provides an alternate approach for the
isolation of DNA from 1 – 10 ml mammalian whole blood, buffy coat, or lymphocytes
...
After all erythrocyte
components are removed, leukocyte DNA can be isolated free of interfering hemoglobin
...
90 min, and can easily process multiple samples
...
The isolated DNA can be used in any application requiring genomic DNA, including
long-template PCR and genomic Southern hybridizations
...
coli, yeast, and mouse
tails
...
Starting material quantities ranging from 100 mg to
1 gram of tissue, or from 1 x 107 to 5 x 107 cultured cells, can be analyzed with the kit
...
TriPure Isolation Reagent
Analysis of gene expression requires clean, intact RNA templates
...
To minimize RNase
activity, RNA isolation procedures typically begin with cell or tissue lysis in a strongly
denaturing environment
...
A simpler alternative, the TriPure Isolation Reagent, offers a rapid RNA isolation
procedure that can easily process multiple samples and produces 30 – 150% more RNA
than other purification methods
...
4
Thus, the TriPure Isolation Reagent allows the simultaneous isolation of RNA, DNA, and
protein from the same sample
...
The
isolated RNA-free DNA may be used for PCR, restriction digest, or Southern blots
...
References
Chomczynski, P
...
A
...
(1989) PCR Technology: Principles and Applications for DNA Amplification, p
...
, Fritsch, E
...
, and Maniatis, T
...
2, p
...
14 – 9
...
Y
...
No
...
The remaining leukocytes
are lysed with a strong anionic detergent, and proteins are removed by dehydration and
precipitation
...
– 10 ml whole blood (research samples) that has been treated with an anticoagulant
1
(sodium heparin, sodium citrate, or EDTA)
The kit works best with fresh blood or blood stored (at +2 to +8°C or –15 to –25°C)
for #3 days
...
Lymphocyte preparations from 10 ml mammalian blood (research samples)
Buffy coat preparations from 10 – 20 ml mammalian blood (research samples)
Application
or preparation of high molecular weight, purified genomic DNA, which may be
F
used in standard and long-template PCR, Southern blots, etc
...
Time required
otal time: approx
...
Purity: Average A260/A280 of isolated DNA = 1
...
9
...
ncreases lab efficiency, because the kit can prepare multiple DNA samples, free of
I
RNA and protein, in approx
...
Increases lab safety, because the kit does not require extensive handling of poten
tially hazardous samples, nor use hazardous organic reagents
...
154
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Mammalian Blood
How to use the kit
How to use the kit
I
...
Kit contents
Red Blood Cell Lysis Buffer (750) ml
White Blood Cell Lysis Buffer (125) ml
Protein Precipitation Solution (65 ml)
III
...
0 (or other suitable DNA storage buffer)
terile centrifuge tubes that can hold at least 40 ml (preferably 50 ml) and withstand
S
a centrifugation force of 900 x g
Sterile 17 x 100 mm tubes that can withstand a centrifugation force of 12,000 x g
RNase (for optional digestion step)
Solution-based Isolation
155
DNA Isolation Kit for Mammalian Blood
How to use the kit
IV
...
7 – 1
...
Starting with blood that had been stored for 7 days at +2 to +8°C or #1 month
at –15 to –25°C will lead to a 10 – 15% reduction in yields
...
Protocol for preparing DNA from 10 ml samples of whole
blood
General notes:
E o prepare DNA from buffy coat, lymphocytes, or blood volumes <10 ml, modify
T
this protocol as detailed in the package insert supplied with the kit
...
E Warm blood samples to +15 to +25°C
...
To each tube:
E Add 10 ml mammalian whole blood
...
Do not vortex tubes
...
E OR inverting the sample at regular intervals by hand, for a total of 10 min
...
Do not centrifuge the tube at more than 875 x g
...
Is it clear and red?
E If yes, red cells have lysed completely
...
E If no, red cells did not lyse
...
Carefully pour off and discard the clear, red supernatant
...
Vortex the tube to thoroughly resuspend the white cell pellet in the residual supernatant
...
E Mix the contents of the tube thoroughly by vortexing
...
E Did the starting sample contain heparin?
E If no, skip this step and go directly to Step 8
...
Do you need the final DNA preparation to be free of RNA?
4
E If no, skip this step and go directly to Step 9
...
02 µg RNase per µl DNA solution
...
E Incubate the tube at +37°C for 15 min
...
For each sample:
E Add 2
...
E Vortex the tube thoroughly (for approximately 25 s)
...
Carefully pour the supernatant, which contains the DNA, into a new sterile 50 ml
centrifuge tube, then:
E Add 2 volumes of absolute ethanol +15 to +25°C
...
E Gently mix the contents of the tube by inversion until the DNA strands precipitate
and the remaining liquid is no longer cloudy
...
E Discard the supernatant
...
Do not vortex
...
EEE
Solution-based Isolation
157
DNA Isolation Kit for Mammalian Blood
How to use the kit
E If you want to collect the DNA by spooling, then:
E Wind the DNA strands around a sterile, blunt-ended glass rod
...
E Swirl the glass rod until the DNA strands are released into the 70% ethanol
...
Centrifuge the tube containing 70% ethanol and DNA for 5 min at 875 x g
...
Dry the DNA pellet by:
E EITHER placing the tube under vacuum without heat until the ethanol is no
longer visible (usually <5 min)
...
To rehydrate and dissolve the DNA pellet:
E Add 1 ml of Tris-EDTA (pH 8
...
E Vortex thoroughly
...
For DNA from human blood, use a 30 min incubation
...
E During the incubation, periodically vortex the tubes to facilitate solubilization of
the DNA
...
E Store DNA at +2 to +8°C
...
Troubleshooting the DNA Isolation protocol
If you get
...
And you should
...
E Use a 15 min incubation in Step 3
...
Particles present
in sample after
vortexing
(Step 7)
Incomplete
white cell lysis
Using fresh samples, repeat Protocol Va, with one or more changes:
E n Step 6, be sure the white cell pellet is fully resuspended in
I
the residual supernatant after vortexing
...
E n Step 7, add enough White Blood Cell Lysis Buffer to ensure
I
the solution is not viscous and does not contain clumps of
cells
...
EEE
158
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Mammalian Blood
How to use the kit
VI
...
Then, the
cause may be
...
Using fresh samples, repeat Protocol Va, with one or more changes:
E n step 10 vortex the tube for approximately 25 s after adding
I
Protein Precipitation Solution
...
Very few leukocytes E o nothing
...
DNA that is
difficult to dissolve
Precipitated
E n Step 16, let the DNA rehydrate and dissolve overnight at
I
DNA was overdried
+2 to +8°C
...
E In Step 7, use less lysis buffer
...
4
E epeat protocol, but use fresh blood or blood that has been
R
stored <3 days at +2 to +8°C or –15 to –25°C
...
present in DNA
(A260/A280 <1
...
present
E Increase the RNase incubation time to 30 min
...
0)
Concentration of
DNA not optimal
for application
E etermine the amount of DNA in the preparation, then use
D
the same amount of DNA in the application that you would
normally use if you prepared DNA by another purification
method
...
Human and mouse blood
research samples were collected as previously described (Noeth and Dasovich-Moody, 1997)
...
Aliquots of each DNA preparation were used as
templates for the long-template PCR amplification of several gene fragments
...
Left panel shows gene fragments amplified from
human DNA:
Lanes 1, 10: DNA Molecular Weight Marker III
Lanes 2, 3: tPA fragment (9
...
2 kb) amplified from 330 ng
DNA
Lanes 4, 5: a-2 collagen fragment (5
...
4 kb)
amplified from 50 ng DNA
Lanes 8, 9: a-2 collagen fragment (15
...
Experiment 2
1
2 3 4
5 6 7 8 9 10 11 12
Figure 47: Use of DNA from various human blood
research samples (prepared with the DNA
Isolation Kit for Mammalian Blood) for detection
of the n-ras gene by Southern hybridization
...
DNA
was also prepared from a lymphocyte preparation
and a buffy coat preparation, using a modification of
the protocol outlined in the kit package insert
...
DNA on the membrane was hybridized to
a DIG-labeled n-ras probe, and the results visualized
chemiluminescently
...
2 kb) size (Taparowsky et
al
...
160
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Cells and Tissues
References
Castellano, M
...
(2006) Ann
...
Y
...
Sci
...
et al
...
J
...
, 161, 495 – 502
Jasik, A
...
(2006) J Vet Diagn Invest, 18, 270 – 274
Kalippke K
...
(2010) Lab Anim
...
et al
...
Ophthalmol
...
Sci
...
and Dasovich-Moody, M
...
-Q
...
(2008) Arterioscler
...
Vasc
...
, 28, 360 – 365
Taparowsky, E
...
(1983) Cell 34, 581 – 586
Uzumcu, A
...
(2006) J
...
Genet
...
et al
...
No
...
This procedure provides a quick, easy, and safe method for
removing contaminating RNA and proteins, resulting in purified genomic DNA ranging
in size from 50 – 150 kb
...
RNA is eliminated with an
RNase treatment and proteins are removed by selective precipitation and centrifugation
...
4
Starting material
100 mg – 1 g tissue (research samples)
1 x 107 to 5 x 107 cultured cells (research samples)
Up to 1011 gram negative bacteria
Up to 3 x 105 yeast cells
50 – 400 mg mouse tails
Application
Isolated DNA is suitable for many applications, including standard PCR, long
template PCR, and Southern blots
...
After quantification we recommend usage of the same amount
of DNA per application as typically used in an alternative purification method
...
5 h (plus resuspension time)
Total time for cultured cells:
adherent cells/trypsinized #4
...
5 h (plus resuspension time)
suspension cells #4
...
The A260/280 ratio for
isolated DNA samples is typically 1
...
9
...
Benefits
Safe, eliminates the need for organic extractions or chaotropic reagents
...
5 hours for
I
tissue (plus resuspension time)
...
All reagents necessary are contained in the kit
...
162
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Cells and Tissues
How to use the kit
How to use the kit
I
...
Kit contents
Cellular Lysis Buffer, (150 ml)
Proteinase K Solution (100 µl)
RNase Solution (5 ml)
Protein Precipitation Solution (60 ml)
III
...
0 (optional)
4
Trypsin (optional)
IV
...
If frozen or refrigerated starting material is used, yields may be reduced
...
E Standard procedure is for a 400 mg sample size
...
Cultured cells
Gram Negative Bacteria
(e
...
, E
...
Yeast
E Count cells and use up to 3 x 105 cells
...
Mouse tail
164
E Procedure is written for the isolation of DNA from 5 x 107
cells
...
E Use 50 – 400 mg mouse tail and follow the procedure titled,
„Isolation of DNA from Mouse Tails“
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Cells and Tissues
How to use the kit
IVa
...
Prior to use, Resuspend the Cellular Lysis Buffer by placing it at +37°C for approximately 5 min
...
Prior to the isolation, count cells via a Coulter Counter or hemocytometer
...
If using fewer cells refer to point IVb
...
Lysis and RNA removal
The following procedure applies to 5 x 107 cells (adherent or suspension)
Homogenize sample until cells are a fine suspension (approximately 10 – 15 s on a
medium setting of a Brinkman Polytron Homogenizer or equivalent)
...
Vortex sample for 2 – 3 s to ensure Proteinase K Solution is mixed with the suspension
...
Remove sample from +65°C, loosen cap to vent
...
Stock concentration of RNase Solution is 10 mg/ml
...
Place sample at +37°C for 15 min
...
Protein precipitation
Add 6 ml Protein Precipitation Solution to each sample
...
Vortexing is necessary for effective removal of protein from the sample
...
This step is important because it aids in precipitation of the protein
...
g
...
Ensure that the tube can withstand a centrifugation of 26,900 x g (rmax)
...
g
...
Samples must be centrifuged at 26,900 x g (rmax) for a minimum of 20 min
...
EEE
Solution-based Isolation
165
DNA Isolation Kit for Cells and Tissues
How to use the kit
Carefully pipette the supernatant containing the DNA into a new, sterile 50 ml
centrifuge tube
...
Pipette from the opposite side of the tube, away from the protein pellet,
to ensure that none of the pellet is pipetted into the sample
...
7 volumes of isopropanol to the sample
...
Usually DNA “strings” will be visible
...
g
...
Discard the supernatant
...
Swirl until
DNA strands are released into the 70% ethanol
...
Save the pellet and add 10 ml cold 70% ethanol to the DNA pellet
...
This will allow the entire pellet to be washed with the 70% ethanol
...
g
...
Discard the supernatant and dry the DNA pellet by placing the sample under vacuum
without heat for a few minutes, or until the ethanol is no longer visible
...
Do not over-dry the DNA pellet as this will make it much more difficult to fully
resuspend the DNA
...
0, or desired buffer
...
Place samples at +50°C for 2 hours to aid resuspension or allow to
resuspend at +2 to +8°C overnight
...
g
...
Store samples at +2 to +8°C until ready to use
...
166
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Cells and Tissues
How to use the kit
IVb
...
E Follow the procedure with the following modification
Cell
Number
Proteinase
Cellular
K Solution
Lysis
Buffer (ml) (µl)
Protein Precipitation Solution (ml)
RNase
Solution Calculate the volume of Protein
Precipitation Solution by multiplying
(µl)
the total volume x 0
...
See example below
...
2
2 x 10
6
4
200
2
...
7
4 x 107
12
8
400
5
7
7
Example:
For 1 x 107 cells, 3
...
4 = 1
...
Cell line/cell type
Average yield per 5 x 107 cells
Range per 5 x 107 cells
CHOK1/Adherent
977
588 – 1522
COS1/Adherent
1984 – 4182
684
463 – 885
PDN-Mouse Hybridoma/
Suspended
Solution-based Isolation
2994
K562/Suspended
1298
1220 – 1487
167
DNA Isolation Kit for Cells and Tissues
How to use the kit
V
...
Then, the
cause may be
...
Protein pellet does
not form, pellet is
soft, or pellet slides
from side of tube
...
Place at +15 to +25°C prior
C
to beginning the procedure
...
Incorrect amount of E lways calculate the amount of buffer needed for each precipiA
precipitation buffer
tation
...
M
E
Place on ice 5 min prior to centrifugation to aid in precipitation
...
addition of
Proteinase K
Solution exceeded
recommended times
DNA does not
precipitate
Incorrect amount of E arefully calculate the amount of isopropanol
...
Sample not mixed
completely
...
C
Discolored DNA
Certain tissues such E ash sample with 70% ethanol soon after isopropanol
W
as liver may discolor
precipitation centrifugation step
...
DNA yield lower
than expected
Incomplete lysis
260/280 ratio
too high (>1
...
E ysis time and buffer volume not correct for sample size
...
e
...
muscle, brain,
heart) may have low
DNA yields due to
difficulty in
processing the
starting material
...
EEE
168
Nucleic Acid Isolation and Purification Manual
DNA Isolation Kit for Cells and Tissues
Typical result with the kit
V
...
Then, the
cause may be
...
260/280 ratio
too low (<1
...
E o not exceed recommended incubation times as this may
D
result in ineffective protein removal from the DNA sample
...
E eat DNA to +65°C for 30 min to aid in resuspension
...
Typical result with the kit
1
2
3 4
5
6
7
8
9
10 11
Figure 48: Amplification of 268 bp to 23 kb
genomic DNA fragments isolated with the
DNA Isolation Kit for Cells and Tissues
...
Lanes 2, 3: Human DMD fragment (268 bp)
and mouse c-myc fragment (580 bp) amplified
using Taq DNA Polymerase
...
2 kb), mouse b2 microglobulin fragment (3
...
9kb),
and human tPA gene fragment (9
...
Lanes 6, 9 and 10: Mouse a-2 collagen gene
fragment (5
...
4) and human bglobin fragment (23 kb) amplified using
Expand Long Template PCR System
...
4
References
Alves-Pereira I
...
(2008) J
...
, 190, 6153 – 6161
Báez, A
...
(2005) Clin
...
11, 3191 – 3197
Hurcombe S
...
A
...
(2009) J Vet Diagn Invest, 21: 266 - 269
Lahiri, D
...
and Schanbel, B
...
Miller, D
...
Dykes and H
...
Polesky (1988) Nucleic Acids Research 16 (3): 1215
...
et al
...
J
...
, 268, 6146
Springer, C
...
(1999) Biochemica 1: 18
Solution-based Isolation
169
TriPure Isolation Reagent
TriPure Isolation Reagent
Clear, red solution; ready-to-use
Cat
...
11 667 157 001 (50 ml)
Cat
...
11 667 165 001 (250 ml)
Principle
During a one-step sample homogenization/lysis procedure, the TriPure Isolation
Reagent disrupts cells and denatures endogenous nucleases
...
The phases
may then be separated and alcohol precipitation used to recover RNA (from the colorless
aqueous phase), DNA and protein (from the interphase and red organic phase)
...
2
...
25 min for RNA isolation
Results
ields vary depending on starting material (See the table under Part IV of “How to
Y
use the reagent” in this article)
...
6 – 2
...
7
Benefits
aves time, because the RNA isolation procedure requires only 1 h
...
Adapts easily to needs of specific laboratories, because the reagent can be used
with a wide variety of starting samples
...
ncreases yield of intact RNA, because the reagent provides an immediate chao
I
tropic denaturing environment that eliminates endogenous RNase activity
...
Flow diagram
(see page 173)
Colorless aqueous phase
containing RNA
White interphase
Red organic phase
Contain DNA
and protein
II
...
It is ready to use as supplied
...
Additional materials needed
For the extraction and phase separation protocol
terile, disposable polypropylene tubes that can withstand 12,000 x g in the presence
S
of TriPure Isolation Reagent and chloroform
hloroform (free of all additives such as isoamyl alcohol)
C
For RNA isolation
sopropanol
I
5% ethanol
7
iethylpyrocarbonate (DEPC)-treated, RNase-free water or DEPC-treated 0
...
1 M sodium citrate in 10 % ethanol
0
For protein isolation
sopropanol
I
% sodium dodecyl sulfate (SDS)
1
...
No
...
Average nucleic acid yield from various sources
Sample
172
DNA yield
Tissue:
Liver
Spleen
Kidney
Skeletal muscle or brain
Placenta
4
RNA yield
6 – 10 µg/mg tissue
6 – 10 µg/mg tissue
3 – 4 µg/mg tissue
1
...
5 µg/mg tissue
1 – 4 µg/mg tissue
3 – 4 µg/mg tissue
not determined
3 – 4 µg/mg tissue
2 – 3 µg/mg tissue
2 – 3 µg/mg tissue
Cultured cells:
Epithelial cells
Fibroblasts
Human, mouse, or rat cells
8 – 15 µg/106 cells
5 – 7 µg/106 cells
not determined
not determined
not determined
5 – 7 µg/106 cells
Nucleic Acid Isolation and Purification Manual
TriPure Isolation Reagent
How to use the reagent
V
...
Extraction
Add 1 ml TriPure Reagent per 50 – 100 mg tissue
Homogenize sample in tissue homogenizer
Incubate 5 min at +15 to +25°C to dissociate nucleoprotein complexes
Add chloroform (0
...
5 ml per 1 ml TriPure
Reagent Mix by inversion
Incubate 5 – 10 min at
+15 to +25°C
Precipitate with ethanol (EtOH)
(0
...
1 M
sodium citrate
(1 ml per 1 ml TriPure Reagent)
Incubate 30 min at +15 to +25°C
with occasional mixing
Precipitate with isopropanol
(1
...
5% SDS
Incubate 10 – 15 min at
+55 to +60°C to resuspend
Repeat wash step 2 x
Resuspend pellet in
0
...
5 – 2
...
0 – 8
...
Troubleshooting the TriPure Isolation protocol
During
If you get
...
And you should
...
Incomplete solubilization of the final E o not let RNA pellet dry
D
RNA pellet
completely, as a dry pellet will be
much less soluble
...
A260/A280 ratio
<1
...
After homogenization, samples were E tore at +15 to +25°C for 5 min
...
Incomplete solubilization of the final E ncrease incubation time to 30
I
RNA pellet
min at +55°C to solubilize RNA
...
E dd TriPure Isolation Reagent
A
directly to cells attached to
culture dish or flask, according to
package insert instructions
...
E ake appropriate precautions to
T
ensure RNase-free environment
...
Starting samples contained organic
solvents (EtOH, DMSO) or strong
buffers; or had an alkaline pH
E arefully remove the upper
C
aqueous phase for subsequent
RNA isolation, making sure to
avoid the interphase/organic
phase
...
Troubleshooting the TriPure Isolation protocol, continued
During
If you get
...
And you should
...
Incomplete solubilization of the final E o not let DNA pellet dry
D
DNA pellet
completely, as a dry pellet will be
much less soluble
...
7
Incomplete removal of phenol from E ncorporate an additional
I
the DNA preparation (during
sodium citrate/ethanol wash step
...
Samples were homogenized with a
high speed homogenizer
Protein
isolation
Low protein
yield
Too much aqueous phase remained
with the interphase and organic
phase
E arefully remove all of the upper
C
aqueous phase prior to isolation
of DNA
...
1 M sodium
citrate
RNA
c
ontamination
E void using power homogenizer
...
E fter adding 1 ml sodium citrate/
A
ethanol for each 1 ml TriPure
Isolation Reagent (required in
the initial homogenization
process) incubate the sample,
with occasional mixing, for 30
min at +15 to +25°C
...
Incomplete solubilization of the final E ncubate sample at +50°C to
I
protein pellet
completely solubilize the protein
...
Protein
d
egradation
Tissues were not immediately
processed or frozen after removal
from animal
Deformed
bands in PAGE
analysis
Protein pellet not washed sufficiently E ncorporate an additional wash
I
step
...
Total RNA was isolated
(by the TriPure Reagent protocol) from the following research samples: 1
...
0 x 107 human white blood cells, 1
...
The
isolated RNA samples were separated electrophoretically on a gel, transferred to a nylon membrane, and hybridized with a 1 kb, digoxigenin-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe
...
The G3PDH probe recognizes a 1
...
25 µg (lanes 4, 7, 10, 13)
...
References
Arslan F
...
(2010) Circulation, 121, 80 – 90
Bakker, O
...
(1993) BioTechniques 15, 532 – 537
Lousse J
...
et al
...
Reprod
...
D
...
(2010) J
...
, 123, 351 – 359
Schummer, B
...
(1998) Technical Tip Biochemica 2, 31 – 33
Uckun F
...
et al
...
Natl
...
Sci
...
et al
...
Antimicrob
...
, 65, 239 – 242
Weingartl H
...
et al
...
Virol
...
et al
...
J
...
Regulatory Intergrative Comp
...
, 298,
R403 – R410
Solution-based Isolation
177
Notes
4
178
Nucleic Acid Isolation and Purification Manual
Affinity Purification
Overview of Affinity Purification
mRNA Capture Kit
mRNA Isolation Kit
RNA/DNA Stabilization Reagent for Blood/Bone Marrow
mRNA Isolation Kit for Blood/Bone Marrow
180
184
189
197
198
5
Overview of Affinity Purification
Overview of Affinity Purification
Affinity purification is a versatile and highly specific technique for the purification of all
classes of biomolecules utilizing differences in biological activities of chemical structures
...
Often a concentrating effect is reached which enables large volumes to be conveniently
processed
...
The mRNA kits rely on base pairing between poly (A+) residues at the 3’end of mRNAs
and the oligo (dT) residues of a biotin-labeled oligo (dT) probe
...
Some of the described kits prepare mRNA directly whereas the other one starts with the
purification of total nucleic acid and subsequent isolation of the mRNA
...
g
...
Or, for
detailed information on the product most relevant to your research, turn to the page that
describes the product in detail:
If you are interested in
See
page
mRNA Capture Kit
poly(A)+ RT-PCR templates from small amounts of
total RNA (up to 40 µg), cultured cells (up to
5 x 105), or tissue (up to 20 mg), and the simultan
eous immobilization in PCR tubes
184
mRNA Isolation Kit
poly(A)+ RNA from larger amounts of total RNA (up
to 2
...
5 – 5
...
Both can easily
process multiple samples simultaneously
...
Both kits depend upon the affinity of the poly (A)+ tail of mRNA for a biotin-labeled
oligo(dT) probe
...
Once formed, the biotinylated dT-A hybrids can be
immobilized on solid surfaces that have been coated with streptavidin, then washed free
of unbound contaminants
...
In fact, the entire
process, from lysate to final PCR can be done in the same tube
...
30 min
...
In the mRNA Isolation Kit, the biotinylated dT-A hybrids are bound to streptavidincoated magnetic particles
...
The mRNA is readily released from the particles and is pure enough for all
downstream applications, including RT-PCR, Northern blotting, Northern ELISA, and
in vitro translation
...
30 min
...
5
Figure 50: Binding of nucleic acids to magnetic glass
particles
...
Electrophoretic analysis of the
eluate (on an agarose gel in the presence of ethidium
bromide) shows that it contains DNA as well as 28S- and
18S-rRNA
...
In the kit, poly(A)+ RNA is isolated from total nucleic acids with
biotin-labeled oligo(dT)20 and streptavidin-coated magnetic particles
...
Stabilization of total nucleic acids followed by mRNA isolation
The approach involves two distinct steps: Whole blood samples or bone marrow
aspirates (research samples) are lysed and instantaneously stabilized with the RNA/DNA
Stabilization Reagent for Blood/Bone Marrow
...
The advantages
of this approach are:
his method involves no cell separation step; thus, there is no danger of losing rare
T
cells that show an aberrant sedimentation or lysis behavior
...
The characteristics of the method is summarized in the following table:
5
Characteristic*
mRNA Isolation Kit for Blood/Bone Marrow
Starting Material
Blood or bone marrow stabilized with RNA/DNA
Stabilization Reagent for Blood/Bone Marrow (up to 5 ml)
Removal of RBCs
Not required
Lysis of WBCs
Not required
Total NA isolation with MGPs
Required
mRNA isolation with SMPs
Required
Final mRNA elution volume
20 µl
* bbreviations: MGPs, magnetic glass particles; NA, nucleic acids; RBCs, red blood cells; SMPs, streptavidinA
coated magnetic particles; WBCs, white blood cells
Advantages of the kit
lexible enough to handle a range of sample sizes
F
ensitive enough to detect rare mRNAs
S
owerful enough to remove inhibitors
P
We estimate that, to obtain a positive RT-PCR from a research blood sample
containing tumor cells (one cell/ml, each with 10 specific mRNA transcripts), you
would need to amplify the mRNA from at least 2 ml of whole blood
...
The
mRNA Isolation Kit for Blood/Bone Marrow was
used to isolate mRNA from different volumes of
heparinized human blood research samples
...
C
Lane A: mRNA from 5 ml blood
Lane B: mRNA from 3 ml blood
Lane C: mRNA from 1
...
There
was no evidence of degradation products in the
preparation
...
5 1
...
Varying amounts of b-actin mRNA (1 ng, panel A; 40 pg, panel B
and 1
...
The mixtures were used as
templates for RT-PCR
...
5 µg rRNA (less than the amount in 1 ml blood) completely inhibited the specific
amplification of b-actin mRNA
...
6 pg
mRNA (equivalent to the mRNA from 1
...
Affinity Purification
183
mRNA Capture Kit
mRNA Capture Kit
for capturing 192 poly(A)+ RNA preparations in PCR tubes
Cat
...
11 787 896 001
Principle
A lysis step releases RNA from cultured cells or tissues
...
A streptavidin-coated PCR tube immobilizes the biotin-labeled dT-A hybrids and washes remove unbound contaminants
...
The entire process (including RT-PCR) takes place in a single tube
...
Starting material
Research samples may contain up to:
µg total RNA
40
5 x 105 cultured cells
20 mg tissue
Application
5
Preparation of highly purified poly(A)+ RNA, which may be used directly for qualitative
or quantitative RT-PCR
Time required
Total time: approx
...
Purity: When isolated mRNA is used as template for RT-PCR, the amplicons are
clearly visible and free of background (See “Typical results with the kit” in this
article)
...
Minimizes sample loss, handling time, and contamination, because the kit allows
rapid, efficient isolation of mRNA and RT-PCR in a single tube
...
184
Nucleic Acid Isolation and Purification Manual
mRNA Capture Kit
How to use the kit
How to use the kit
I
...
Kit contents
Lysis Buffer containing lithium dodecyl sulfate (50 ml)
Oligo(dT)20 Concentrate: Oligo(dT)20, biotin-labeled, 20x concentrated (50 µl)
Dilute the Oligo(dT)20 Concentrate just before use
...
Additional materials needed
Sterile cups (for preparing dilutions)
Pipettes with sterile, aerosol-resistant pipette tips
Syringe fitted with a 21-gauge needle (for tissue and cultured cells only)
PBS (for preparing cultured cells only)
Mortar, pestle, liquid nitrogen (for preparing tissue samples only)
PCR thermal cycler
Reagents for RT-PCR
Electrophoresis equipment or other equipment for analyzing PCR products
IV
...
3 – 25
Tissue (100 mg):
Mouse brain
Mouse liver
Mouse lung
200
700
130
7
14
10
7
5
V
...
M
For decontamination of equipment, see Appendix (page 219)
...
To prepare the Oligo(dT)20 Working Solution used in Step 3 below, dilute the
Oligo(dT)20 Concentrate 1:20 with PCR grade water
...
(Each capture requires 4 µl of Working Solution
...
E Cultured cells: Wash cells (5 x 105) twice with ice-cold phosphate-buffered
saline (PBS)
...
Shear DNA mechanically
by passing the sample 6 x through a 21-gauge needle
...
Grind frozen tissue to a homogeneous
powder in a pre-cooled mortar
...
Homogenize the powder suspension by passing it 4 x through a 21-gauge needle
...
Use only the supernatant for the
capture procedure
...
Otherwise, perform these steps at +4°C
...
E Incubate the hybridization mix for:
E EITHER 5 min at +2 to +8°C (if you started with tissue)
E OR 5 min at +37°C (if you started with cultured cells or total RNA)
Immobilize mRNA in the Streptavidin-coated PCR Tubes:
E Add 50 µl of the hybridization mix to a Streptavidin-coated PCR tube
...
E Incubate the hybridization mix for:
E EITHER 3 min at +2 to +8°C (if you started with tissue)
E OR 3 min at +37°C (if you started with cultured cells or total RNA)
Wash the immobilized sample 3 times
...
To avoid losing mRNA, do not wash the sample too vigorously
...
After the third wash, the captured mRNA in the Streptavidin-coated PCR
Tube is ready for RT-PCR
...
Troubleshooting the mRNA Capture protocol
If you get
...
And you should
...
material
mRNA trapped inside sample clumps can not be isolated by
this procedure
...
of Lysis Buffer
E ilute the lysate (Step 1) before continuing the procedure
...
Degradation of
RNA by contaminating RNase from
buffers or equipment
Degradation of
RNA because
RNases in sample
material were not
sufficiently inactivated
E heck all buffers for RNase contamination
...
E ollow the kit procedure exactly as written and be especially
F
careful to:
E ork rapidly
W
E erform indicated steps at ice water temperatures
P
(0 to –4°C)
Affinity Purification
187
mRNA Capture Kit
Typical result with the kit
Typical result with the kit
MCAD
b-Actin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 53: Detection of MCAD and b-actin transcripts in K562 cells
...
Messenger RNA from the dilutions was captured in Streptavidin-coated PCR tubes according to the
above protocol
...
Amplicons were run on a 1% agarose gel
...
5
Lanes 1, 10, 18: DNA Molecular Weight Marker VI
The remaining lanes contained mRNA from
Lane 2: 1 x 104 cells
Lanes 3, 11: 2 x 103 cells
Lanes 4, 12: 400 cells
Lanes 5, 13: 80 cells
Lanes 6, 14: 16 cells
Lanes 7, 15: 3
...
64 cells
Lanes 9, 17: Control (no RT performed)
Result: The mRNA Capture Kit and Titan One Tube RT-PCR System allow the efficient detection of b-actin and
MCAD template RNA’s from as few as 3
...
References
Bax M
...
(2007) J
...
, 179, 8216 – 8224
Blümel, J
...
(2005) J
...
, 79, 14197 – 14206
Breiner, H
...
et al
...
et al
...
Immunol
...
et al
...
, 67, 3919 – 3926
Cote, G
...
(1997) Biochemica 4, 14 – 18
de Jong M
...
W
...
et al
...
Gen
...
, 89, 2398 – 2409
Feron, R
...
(2004) Biochemica 3: 23 – 24
Fillingham, J
...
et al
...
Cell, 5, 1347 – 1359
García-Vallejo, J
...
et al
...
Biol
...
, 280, 12676 – 12682
Geurtsen J
...
(2009) J
...
, 183, 5221 – 5231
Marein A
...
P
...
(2010) J
...
, 185: 1633 - 1641
Tian C et al
...
Immunol
...
No
...
Streptavidincoated magnetic particles capture the biotinylated dT-A hybrids
...
Then, water
elutes the mRNA from the particles
...
5 mg total RNA
108 cultured cells
1 g tissue
Application
Preparation of highly purified poly(A)+ RNA, which may be used for RT-PCR, cDNA
synthesis, Northern blotting, Northern ELISA, RNase protection assays, in vitro translation, etc
...
30 min (starting from total RNA)
Hands-on time: approx
...
urity: Isolated mRNA, free of DNA and other RNAs
...
5
ccommodates a wide range of samples, and allows both small and large scale
A
preparations of mRNA
...
Affinity Purification
189
mRNA Isolation Kit
How to use the kit
How to use the kit
I
...
Kit contents
Lysis Buffer containing lithium dodecyl sulfate (100 ml)
Streptavidin-coated Magnetic Particles (1
...
Additional materials needed
Magnetic Particle Separator
Sterile tubes and cups
Pipettes with sterile, disposable pipette tips
Syringe fitted with a 21-gauge needle (for tissue and cultured cells only)
PBS (for preparing cultured cells only)
Mortar, pestle, liquid nitrogen (for preparing tissue samples only)
5
IV
...
3 - 25
Tissue (100 mg):
Mouse brain
Mouse liver
Mouse lung
200
700
130
7
14
10
7
V
...
For decontamination of equipment, see Appendix (page 219)
...
To isolate mRNA from different amounts or volumes of sample, you must
alter the amounts of reagents used in the procedures below
...
Prepare the sample:
E Total RNA: Dilute 500 µg total RNA (up to 200 µl) with Lysis Buffer (final total
volume, 400 µl)
...
Do not dilute the Lysis Buffer more than twofold
...
Add 3 ml Lysis Buffer to the cell pellet
...
E Tissue: Snap freeze 200 mg of tissue
...
Chill 3 ml Lysis Buffer to 0°C in a sodium
chloride-ice water bath, then add the frozen powder to the chilled Lysis Buffer
...
Centrifuge the suspension at 11,000 x g for 30 s
...
Perform all steps in the capture procedure below at a temperature between
0°C and –4°C
...
E Immobilize the Streptavidin-coated Magnetic Particles on the side of the container
with a Magnetic Particle Separator
...
E Resuspend the Streptavidin-coated Magnetic Particles in 500 µl Lysis Buffer
...
5
Never let the Streptavidin-coated Magnetic Particles dry out
...
Immobilize the biotinylated dT-A hybrids with Streptavidin-coated Magnetic Particles:
E Add hybridization mix to the tube containing the prepared Streptavidin-coated
Magnetic Particles (from Step 2)
...
E Incubate for:
E EITHER 5 min at +37°C (if you started with total RNA or cultured cells)
...
Separate the Streptavidin-coated Magnetic Particles from the fluid with a Magnetic
Particle Separator
...
Wash the Streptavidin-coated Magnetic Particles 3 times
...
E Resuspend the Streptavidin-coated Magnetic Particles in the Wash Buffer
...
E Remove all Wash Buffer and discard
...
E Resuspend the Streptavidin-coated Magnetic Particles in the redistilled water
...
E Separate Streptavidin-coated Magnetic Particles from the eluate with a Magnetic
Particle Separator
...
To quantitate the mRNA spectrophotometrically, assume that 1 A260 unit
corresponds to a concentration of 40 µg/ml
...
Troubleshooting the mRNA Isolation protocol
If you get
...
And you should
...
mRNA trapped inside sample clumps cannot be isolated by
this procedure
...
of Lysis Buffer
The viscosity of the lysate is critical to successful mRNA
(lysate viscosity too
isolation
...
Elution volume too E Repeat elution
...
Wrong lysis
buffer or lysis
buffer with too
much guanidine
thiocyanate (GTC)
E Use only the Lysis Buffer provided in the kit
...
E f you use a lysis buffer containing GTC, follow the guidelines
I
in the package insert for GTC concentration and hybridization
temperature
...
RNase contamination in buffers
5
E Check all buffers for RNase contamination
...
g
...
E ollow published procedures (e
...
, Farrel, 1983) for RNase
F
decontamination
...
of lysis buffer
E se the isolated mRNA as starting material and repeat the
U
(lysate viscosity too
isolation protocol
...
mRNA was isolated from liver tissue according to the above
protocol
...
The hybrids were visualized with an enzyme-labeled anti-DIG
antibody and a chemiluminescent enzyme substrate
...
No
degradation products were visible
...
[submitted by T
...
Wheeler-Schilling, M
...
Guenther, and K
...
Specifically, attention
focused on the renin-angiotensin system (RAS), in which angiotensinogen is cleaved by
renin and angiotensin-converting enzyme (ACE) to produce the active peptide angiotensin II (Oparil and Haber, 1974)
...
However,
increasing evidence suggests that an RAS is expressed locally in various tissues
...
, 1991)
...
To obtain enough mRNA template from these neuronal tissues, we
used the mRNA Isolation Kit
...
The eyes of each chicken (Gallus
domesticus, 12 days old) were dissected immediately after enucleation by hemisection
along the ora serrata
...
Tissues were homogenized
and mRNA isolated with the mRNA Isolation Kit, according to the protocol given in the
kit package insert
...
Isolated mRNA samples were treated with RNase-free DNase,
extracted with phenol, and precipitated with ethanol, according to standard procedures
...
194
Nucleic Acid Isolation and Purification Manual
mRNA Isolation Kit
Typical results with the kit
ACE-specific PCR
...
The entire product (20 µl) of the reverse transcription reaction was amplified in a
100 µl reaction mix containing 10 mM Tris HCl (pH 8
...
5 mM MgCl2,
3% dimethylsulfoxide, 10 mM dNTPs, 15 pmol of each ACE-specific primer, and 2
...
The sequences of the ACE-specific primers were deduced from
EMBL; AC: L40175 (Esther et al
...
The final extension was 10 min at +72°C
...
e
...
7 – 9
...
The yield was
highly reproducible (data not shown)
...
The specificity of the amplified sequence was tested by digestion of the
amplicon with Pvu II
...
These results indicate that ACE gene expression occurs in the retina, choroid, pecten,
optic nerve and iris/ciliary body of the chicken eye
...
5
Figure 55: Detection of ACE-specific mRNA in chicken eye tissue by RT-PCR
...
The mRNA was amplified by RT-PCR,
using primers derived from the gene for angiotensin converting enzyme (ACE)
...
5% agarose gel and stained with ethidium bromide
...
Pvu II split the amplicon into two fragments
of 388 bp and 137 bp
...
(1998) Biochemica Information 102, 30 – 31
Botelho R
...
et al
...
Biol
...
, 284, 28522 - 28532
Bönsch C et al
...
Virol
...
et al
...
Virol
...
E
...
Academic Press, New York
Guo L
...
et al
...
Acids Res
...
et al
...
Biol
...
, 283, 25475 – 25484
Sambrook, J
...
F
...
(1989) Molecular Cloning: A Laboratory
Manual
...
et al
...
, 20: 1432 - 1440
5
196
Nucleic Acid Isolation and Purification Manual
RNA/DNA Stabilization Reagent for Blood/Bone Marrow
RNA/DNA Stabilization Reagent for
Blood/Bone Marrow
for cell lysis and stabilization of nucleic acids contained in blood or bone marrow
Cat
...
11 934 317 001
Principle
The purification of mRNA from blood or bone marrow requires two steps:
1
...
The Stabilization Reagent inhibits nuclease activity to protect nucleic acids
in the lysate from degradation
...
fter lysis, total nucleic acids are purified with the mRNA Isolation Kit for Blood/Bone
A
Marrow
...
The mRNA Isolation Kit for Blood/Bone Marrow,
described on the following page, is used only for Step 2 of the procedure
...
5 – 5
...
Application
apid stabilization of nucleic acids from human whole blood or bone marrow
R
research samples
Time required
Total time: <5 min (for lysis of sample and stabilization of nucleic acids in lysate)
5
Hands-on time: <5 min
Results
ucleic acids stabilized with the RNA/DNA Stabilization Reagent for Blood/Bone
N
Marrow may be stored up to 1 year at –15 to –25°C, 1 day at +2 to +8°C, or 6 h at
+20 to +25°C
...
Protects nucleic acids in the lysed sample from degradation by nucleases
...
No
...
Cells from fresh blood or bone marrow are lysed with the RNA/DNA Stabilization
Reagent for Blood/Bone Marrow
...
fter lysis, total nucleic acids released from blood or bone marrow are bound to
A
Magnetic Glass Particles, washed free of cellular contaminants, then released from the
Magnetic Glass Particles
...
The mRNA is bound to the
Oligo(dT)-Streptavidin-coated Magnetic Particle complex, while other nucleic acids
(DNA, rRNA, tRNA) are washed away
...
5
The RNA/DNA Stabilization Reagent for Blood/Bone Marrow, described on the previous
page, is used only for Step 1 of the procedure
...
Starting material
ysates containing nucleic acids from 1
...
0 ml human blood or bone marrow
L
aspirates (research samples), obtained and stabilized with the RNA/DNA Stabilization Reagent for Blood/Bone Marrow
Application
solation of highly purified, human mRNA, which is suitable for RT-PCR, cDNA
I
synthesis, nuclease protection assays, Northern blotting, or Northern ELISA
Time required
otal time: 1
...
1 h
H
Results
ypical mRNA yield: approx
...
T
Since white blood cell count can differ significantly between donors, mRNA yield
can range from 50 to 200 ng/ml blood
...
V
ensitivity: In a model system, it was possible to detect melanoma mRNA (by RTS
PCR) in a kit-prepared sample containing mRNA from 5 ml of fresh blood that had
been spiked with mRNA from 5 melanoma cells (Mel-Ju)
...
ncreases the efficiency of RT-PCR screening by removing PCR inhibitors (rRNA,
I
tRNA, and hemoglobin) from the mRNA template
...
A
roduces high yields of purified, intact mRNA, suitable for multiple research
P
applications
...
I
198
Nucleic Acid Isolation and Purification Manual
mRNA Isolation Kit for Blood/Bone Marrow
How to use the reagent and the kit
How to use the reagent and the kit
I
...
Lyse red blood cells and stabilize total nucleic acids
with RNA/DNA Stabilization Reagent for Blood/Bone
Marrow (separate reagent; not contained in kit)
2
...
Wash the Magnetic Glass Particles 4 times and collect
them with a Magnetic Particle Separator after each
wash
4
...
Immobilize mRNA with biotinylated Oligo(dT) and
Streptavidin Magnetic Particles
5
6
...
Elute mRNA from Streptavidin-coated Magnetic
Particles
RNA/DNA Stabilization Reagent
for Blood/Bone Marrow is not
contained in the kit
Affinity Purification
199
mRNA Isolation Kit for Blood/Bone Marrow
How to use the reagent and the kit
II
...
agnetic Glass Particles Elution Buffer (33 ml)
M
ybridization Buffer (17 ml)
H
ligo(dT)20 Probe, biotin-labeled (330 µl)
O
treptavidin-coated Magnetic Particles (3
...
Additional materials needed
NA/DNA Stabilization Reagent for Blood/Bone Marrow, with guanidine isothiocyaR
nate and Triton X-100 (500 ml)
5
The reagent crystallizes at temperatures below +20°C
...
Thoroughly mix before use
...
Protocols for preparing mRNA from 5 ml of human whole
blood or bone marrow research samples
When handling blood, bone marrow, and blood/bone marrow lysates, take
the precautions you usually take when handling potentially hazardous
material
...
IVa
...
Label the tube with all relevant information
(identification, kind of sample, total volume, date and time of draw, delay between draw
and processing)
...
E Add 5 ml of sample
...
Do one of the following:
5
E If you are going to isolate mRNA immediately, go to Procedure IVb
...
Between now and the start of Procedure IVb, do one of the following:
E Store lysate as follows:
E Up to 1 year at –15 to –25°C
E 1 day at +2 to +8°C
...
E Place the lysate on dry ice for transport to another facility
...
Isolation of mRNA from 55 ml of lysate (equivalent to 5 ml of
sample)
For preparation of mRNA from small samples (1
...
Sample preparation: All lysates should be at +20 to +25°C
...
Thoroughly mix (e
...
, by vortexing) to ensure crystallized material is fully dissolved
...
In a sterile, screw-cap 50 ml centrifuge tube:
E Add entire sample lysate from Procedure IVa
...
E Vortex for 10 s
...
EEE
Affinity Purification
201
mRNA Isolation Kit for Blood/Bone Marrow
How to use the reagent and the kit
After the incubation:
E Centrifuge the tube for 2 min at 1100 x g and +15 to +25°C
...
E Invert the tube and place on filter paper for 30 s
...
Transfer the Magnetic Glass Particles to a 2 ml microcentrifuge tube, then wash them
4 times
...
E Collect the Magnetic Glass Particles on the side of the tube with a Magnetic
Particle Separator
...
E Wait a few seconds, then remove (and discard) any residual supernatant
...
E Incubate suspension for 5 min on a 1400 rpm shaker at +70°C
...
E Immediately transfer the supernatant to a fresh 2 ml tube
...
E Immediately transfer the supernatant to a fresh 2 ml tube
...
To isolate mRNA from the total nucleic acids:
E Prepare 100 µl Streptavidin Magnetic Particles: Transfer 100 µl SMP suspension to
a fresh 2 ml tube
...
Withdraw and
discard the supernatant (storage solution) from the tube
...
5 ml Hybridization Reagent: Add 10 µl biotin-labeled Oligo(dT)20 Probe to
0
...
E Add 0
...
Incubate for 2 min at +37°C
...
E Let the tube stand for 5 min at +37°C
...
E Discard the supernatant
...
For each wash:
E Resuspend Streptavidin Magnetic Particles in 0
...
E Collect the Streptavidin Magnetic Particles on the side of the tube with a Magnetic
Particle Separator
...
EEE
202
Nucleic Acid Isolation and Purification Manual
mRNA Isolation Kit for Blood/Bone Marrow
How to use the reagent and the kit
To elute the mRNA from the Streptavidin Magnetic Particles:
E Resuspend Streptavidin Magnetic Particles in 20 µl PCR grade water with a
pipette
...
E Collect the Streptavidin Magnetic Particles on the side of the tube with a Magnetic
Particle Separator
...
E Again use a Magnetic Particle Separator to remove any residual Streptavidin
Magnetic Particles from the supernatant
...
The supernatant (from Step 8) contains the mRNA from the sample
...
E OR store the isolated mRNA at —15 to —25°C or —80°C
...
Troubleshooting the Stabilization and the mRNA Isolation
protocol
If you get
...
Low yield of mRNA RNase contamina(<50 ng/ml
tion in reagents or
blood)
equipment
5
And you should
...
Improper storage of For optimal mRNA stability, store lysates:
lysate before mRNA
E 1 year at –15 to –25°C
isolation
E 1 day at +2 to +8°C
E 6 h at +20 to +25°C
Improper storage of
blood before
stabilization
Fragmented mRNA RNase contamination in reagents or
equipment
Improper
storage of lysate
before mRNA
isolation
Add RNA/DNA Stabilization Reagent for Blood/Bone Marrow
to blood within a few hours after drawing
...
For optimal mRNA stability, store lysates:
E 1 year at –15 to –25°C
E 1 day at +2 to +8°C
E 6 h at +20 to +25°C
Affinity Purification
203
mRNA Isolation Kit for Blood/Bone Marrow
Typical results with the kit
Typical results with the kit
Experiment 1
A
B
C
b-actin
5
204
Figure 56: Stability of mRNA after lysis in RNA/DNA Stabilization Reagent for Blood/Bone Marrow
...
The lysate was divided into three aliquots
...
The isolated mRNA was separated electrophoretically, transferred to a membrane by Northern
blotting, and analyzed with a DIG-labeled antisense b-actin RNA probe
...
However, the sample stored at –15 to –25°C (lanes C)
contained more of the band than the sample stored at +2 to +8°C (lanes A), which in turn contained more than
the sample stored at +15 to +25°C (lanes B)
...
Normal human heparinized bone marrow
and blood were lysed with the RNA/DNA Stabilization Reagent
for Blood/Bone Marrow
...
The mRNA
from 0
...
0 ml of blood
(Lane B) was separated electrophoretically, transferred to a
membrane by Northern blotting, and analyzed with DIG-labeled
antisense b-actin RNA probe
...
Reference
Gault, J
...
(2005) Stroke, 36, 872 – 874
Hansen, I
...
et al
...
et al
...
Endocrinol
...
et al
...
et al
...
, 35, 1559 - 1566
Lukaszewski R
...
et al
...
Vaccine Immunol
...
et al
...
et al
...
et al
...
Or, for
detailed information on the product most relevant to your research, turn to the page that
describes the product in detail:
If you are interested in
Labeled DNA or RNA, free of nucleotides and other
small molecules
210
mini Quick Spin
Columns
208
See
page
Quick Spin Columns
6
For preparing
Labeled DNA, RNA or oligonucleotides, free of
nucleotides and other small molecules
214
Principle of gel filtration chromatography
All the products described in this chapter depend on gel filtration chromatography,
which separates molecules based on their relative size
...
If a molecule can enter and exit the pores of the gel matrix, its rate of movement is
determined by the flow of the chromatographic buffer and the diffusion properties of
the molecule
...
Smaller molecules enter
and leave many pores of the matrix, thus traversing the length of the column relatively
slowly
...
Nucleic Acid Isolation and Purification Manual
Overview of Gel Filtration
Overview of the Quick Spin method
Roche Applied Science has adapted gel filtration technology to a spin column format for
use in a wide range of applications, including removal of:
Unincorporated nucleotides from labeling reactions
Fluorescent dye-labeled dideoxy terminators
...
Fast
Quick Spin Columns for radiolabeled DNA or RNA purification can separate
unincorporated radionucleotides from labeled DNA or RNA in approximately 6 min
...
Longer than most spin columns
Longer columns provide more effective separation, leading to higher yields of DNA
or RNA, with fewer contaminating nucleotides, linkers or primers
...
Reference
Porath, J
...
(1959) Nature 183, 1657 – 1659
Gel Filtration
6
209
Quick Spin Columns
Quick Spin Columns
G-25 or G-50 Sephadex Columns for Radiolabeled DNA
Purification
G-25 or G-50 Sephadex Columns for Radiolabeled RNA
Purification
Cat
...
11 273 922 001
Cat
...
11 273 949 001
20 columns for DNA purification (Sephadex G-25)
50 columns for DNA purification (Sephadex G-25)
Cat
...
11 273 965 001
Cat
...
11 273 973 001
20 columns for DNA purification (Sephadex G-50)
50 columns for DNA purification (Sephadex G-50)
Cat
...
11 273 990 001
20 columns for RNA purification (Sephadex G-25)
Cat
...
11 274 015 001
20 columns for RNA purification (Sephadex G-50)
Principle
Quick Spin columns contain gel filtration matrices which allow large molecules (e
...
,
DNA or RNA) to pass through quickly while retaining small molecules (e
...
, nucleotides)
...
Starting material
Nucleic acids from labeling reactions
Application
Quick and efficient removal of non-incorporated precursors from:
6
Time required
abeled DNA (from nick translation, end-labeling, polymerization, and other
L
labeling reactions)
Labeled RNA (from end-labeling or polymerization reactions)
Total time: 8 min
Hands-on time: 2 min
Results
G-50 Sephadex Columns
xclusion limit:
E
DNA: 72 bp / RNA: 72 bp
etention of nucleotides:
R
DNA: >99 % / RNA: >99 %
ecovery:
R
DNA: 90 % / RNA: 80 %
ample volume:
S
DNA: up to 100 µl / RNA: up to 100 µl
G-25 Sephadex Columns
xclusion limit:
E
DNA: 10 – 12 bp / RNA: 10 – 12 bp
ecovery:
R
DNA: 80 % / RNA: 80 %
Benefits
etention of nucleotides:
R
DNA: 95 % / RNA: 95 %
ample volume:
S
DNA: up to 50 µl / RNA: up to 50 µl
aves time and effort, because columns are ready-to-use
...
ives reproducible results, because columns are quality tested for:
G
aximum retention of unincorporated nucleotides
...
A
210
igh recovery of radiolabeled DNA, RNA, and poly(A)
...
A
Nucleic Acid Isolation and Purification Manual
Quick Spin Columns
How to use the columns
How to use the columns
I
...
Package contents
ach column contains 0
...
0; 1 mM EDTA, 100 mM NaCl)
...
The Quick Spin columns for radiolabeled RNA purification are packed in ziplock
bags to prevent contamination
...
Additional materials needed
abletop or low-speed floor model centrifuge
T
winging-bucket rotor
S
Gel Filtration
211
Quick Spin Columns
How to use the columns
IV
...
Remove most of the buffer from the column as follows:
Remove the top cap from the column, then remove the bottom tip
...
Remove residual buffer from the column as follows:
Place the column in a collection tube
...
Discard the collection tube with the eluted buffer
...
Apply 20 – 50 µl sample to a G-25 column; 20 – 100 µl to a G-50 column
...
Centrifuge the tube at 1100 x g for 4 min in a swinging-bucket rotor
...
It contains the purified sample
...
V
...
Then, the
cause may be
...
Poor sample
recovery
Tipping of the
eep the column upright during and after application of
K
column, which
sample, especially after centrifugation
...
during centrifuga Be sure the centrifuge is correctly calibrated
...
A fixed-angle rotor will cause lower yields
...
02 – 1
...
At
A
>1
...
At <0
...
o improve recovery of dilute samples, add carrier (glycogen,
T
tRNA or sperm DNA) to the sample
...
Troubleshooting the Quick Spin protocol, continued
If you get
...
And you should
...
Column overloaded, Apply a sample containing 0
...
0 mg/ml nucleic acid
...
through the matrix
Wrong g-force
during centrifugation
Use 1100 x g for centrifugation spins
...
Be sure the centrifuge is correctly calibrated
...
Reference
Hiroi M
...
(2010) Am
...
Physiol
...
Physiol
...
et al
...
Cell, 9, 59 – 73
Ramírez M
...
(2009) Eukaryot
...
-C
...
et al
...
Bacteriol
...
et al
...
Endocrinol
...
A
...
152, 25 – 29
Gel Filtration
6
213
mini Quick Spin Columns
mini Quick Spin Columns
Ready-to-use, microcentrifuge-compatible chromatography columns for quick and
efficient purification of nucleic acids from labeling reactions
Cat
...
11 814 419 001
Cat
...
11 814 427 001
Cat
...
11 814 397 001
50 mini Quick Spin DNA Columns
50 mini Quick Spin RNA Columns
50 mini Quick Spin Oligo Columns
Principle
The method uses gel filtration chromatography, which separates molecules based upon
their relative size
...
The rapid separation of larger
from smaller molecules may be performed in a conventional tabletop microcentrifuge
...
g
...
Specifically:
se mini Quick Spin DNA Columns to purify radiolabeled or fluorescent dye-labeled
U
DNA ($20 bp) with $90 % recovery
6
se mini Quick Spin RNA Columns to purify radiolabeled RNA ($20 bases) with
U
$80 % recovery
se mini Quick Spin Oligo Columns to purify radiolabeled oligonucleotides
U
($8 bases) with $80 % recovery
Time required
Hands-on time: 2 min
Total time: 7 min (additional 1 min if buffer exchange is needed)
Results
Removal of unincorporated nucleotides
mini Quick Spin DNA or RNA Column $99 % retention
mini Quick Spin Oligo Column $90 % retention
Recovery of labeled probes
mini Quick Spin DNA Columns $90 %
mini Quick Spin RNA/Oligo Columns $80 %
Exclusion limit
mini Quick Spin DNA/RNA Columns $20 bp
mini Quick Spin Oligo Columns $8 bases
Benefits
aves time and effort, because columns are ready to use with any standard microS
centrifuge
...
ives reproducible results, because columns are quality tested to ensure high
G
recovery of labeled DNA, RNA and oligonucleotides with a maximum retention of
unincorporated nucleotides
...
Flow diagram
The following flow diagram summarizes the steps for preparing any mini Quick Spin
Column and purifying a nucleic acid sample with the column
...
Recover eluate containing
the nucleic acids
Additional materials needed
Variable speed microcentrifuge
1
...
Preparing the column
Use the following procedure to prepare any mini Quick Spin Column
...
or
Vortex gently for 3 – 5 s at low speed
...
Excessive vortexing may crush the matrix and lead to contamination on the purified sample with unincorporated nucleotides
...
If the cap is filled with Sephadex, put the cap back on the column and remix
column contents (as in step 1) until most of the matrix is in the body of the
column rather than in the cap
...
EEE
Gel Filtration
215
mini Quick Spin Columns
How to use the columns
Remove excess buffer and pack the column as follows:
Place column in a sterile 1
...
Place the tube in a microcentrifuge rotor
...
(There is a v-shaped notch in the
support ring of the column to help align the column with the rotor
...
Discard the collection tube with the eluted buffer
...
Is the isolated nucleic acid to be used in a fluorescent sequencing reaction?
If no, then skip this step and go to step 5
...
5 ml microcentrifuge tube
...
Centrifuge the tube at 1000 x g for 2 min at +15 to +25°C
...
Go to step 5
...
Minimal salt in the final product means that, when
concentrated, the sample will run cleanly in sequencing applications
...
Delay will allow the column matrix to dry out
...
IV
...
While keeping the column upright, do the following:
Place the prepared column in a clean, sterile 1
...
Very slowly and carefully apply the sample to the center of the column bed
...
Any sample on the side of
the column will bypass the separation matrix and will arrive in the collection tube without being fractionated
...
Do not overload the
column
...
Save the eluate in the second collection tube
...
Discard the mini Quick Spin Column in an appropriate waste receptacle
...
Troubleshooting the mini Quick Spin protocol
This table describes various problems that may occur during the purification procedures
and recommendations for avoiding them
...
Then, the
cause may be
...
Dilution of
final sample
Excess packing
buffer wasn’t
removed before
sample application
efore applying sample, centrifuge column at 1000 x g for
B
1 min to pack the matrix
...
Purified
nucleic acid
contaminated with
unincorporated
nucleotides
To eliminate any liquid remaining atop the column after the
first spin, perform an additional 1 min spin at 1000 x g
...
A
sides of column,
allowing molecules
to flow around,
rather than through
the matrix (without
purification)
Column overloaded o not apply more than the maximum recommended sample
D
volume
...
causing column
matrix to collapse
and unincorporated
nucleotides to pass
freely through
column
6
Column was
o not vortex the column for longer than 5 s
...
Do not use medium or
V
too vigorously
high speed
...
o fully resuspend the matrix before packing step, do one of
T
the following:
nvert column vigorously several times and flick the
I
column sharply to help resuspend the matrix
...
V
Sample volume too Do one of the following:
small (<20 µl)
Add 1x STE buffer to sample until the total sample volume
is 20 µl
...
A
Total volume applied (sample + STE buffer) MUST NOT be
greater than the maximum sample volume recommended
for the column
...
Various oligomers, ranging in size from 32 bp to 8 bp, were
end-labeled using the 5’ End-Labeling Kit and g32P-ATP and purified using either the mini Quick Spin DNA or
Oligo Columns to remove unincorporated nucleotide, per manufacturer’s instructions
...
The gel was
overlaid with plastic wrap and exposed to Lumi-Film for approximately 18 h
...
et al
...
, 38, 117 – 130
Chevance, F
...
V
...
(2006) J
...
, 188, 6561 – 6571
Gajendran N
...
(2009) J
...
, 29, 2404 – 2413
Hawkins B
...
et al
...
Cell
...
, 27, 7582 – 7593
Kitiphongspattana, K
...
(2007) Am J Physiol Endocrinol Metab, 292, E 1543 – 54
Kokay, I
...
et al
...
G
...
E
...
Virol
...
(1999) Biochemica 1, 16 – 17
Safi A
...
(2008) Mol
...
Biol
...
A
...
(2007) J
...
Chem
...
A
...
(2009) J
...
Chem
...
A
...
152, 25 – 29
218
Nucleic Acid Isolation and Purification Manual
Automated Nucleic Acid
Isolation
MagNA Lyser Instrument
MagNA Pure LC Instrument
MagNA Pure Compact Instrument
220
222
223
7
Automated Sample Preparation
Automated Sample Preparation
PCR and real-time PCR application require precise and reproducible nucleic acid
purification
...
The instrument facilitates the production of a supernatant
containing nucleic acids and proteins suitable for subsequent purification, extraction, or
analysis
...
Efficiently homogenize a wide variety of sample materials
...
Prevent nucleic acid degradation with the benchtop cooling block
...
Automate with an easy-to-use instrument
...
Save valuable lab space with a small benchtop instrument
educe hands-on time by replacing the mortar and pestle and other manual
R
m
ethods
...
220
Nucleic Acid Isolation and Purification Manual
MagNA Lyser Instrument
MagNA Lyser Workflow
During a MagNA Lyser Instrument run, the rotor, which is filled with special tubes,
rapidly oscillates
...
e
...
The cells in the sample tubes are disrupted nearly instantaneously when they collide with
the ceramic and glass beads
...
By varying both of these para
meters, optimal disruption of a wide variety of cells can be ensured
...
A
dd your sample and
lysis buffer to the
MagNA Lyser Green
Beads
H
omogenize with the
MagNA Lyser Instrument
Centrifuge to
pellet the
debris
Use the super
natant to
prepare nucleic
acids or proteins
For detailed information, visit www
...
com or contact your local representative
...
The core of the system is a
reliable robotic system, processing up to 32 different samples in one batch
...
User-friendly software protocols adapt to specific sample requirements
...
utomatically isolate any type of nucleic acid
A
The magnetic bead technology used in the MagNA Pure LC System, combined with
dedicated kits, enables consistent isolation of high-quality DNA, RNA, or mRNA for
all demanding nucleic acid research applications
...
xperience fast isolation and easy setup
E
Simple, software-guided handling steps launch the automated isolation procedure,
processing up to 32 samples in as little as 60 minutes
...
et the instrument set up your experiment
L
Following nucleic acid isolation, the unique, programmable post-elution unit of the
MagNA Pure LC Instrument automatically pipets sample and reagents for downstream PCR reactions, for example, with the LightCycler® System
...
Nucleic Acid Isolation and Purification Manual
MagNA Pure Compact Instrument
MagNA Pure Compact Instrument
The MagNA Pure Compact System is the automated benchtop solution for nucleic acid
purification
...
onserve valuable laboratory space with the small footprint
C
btain high-quality nucleic acids
O
from diverse sample types with proven reagent chemistry
...
Save time through easy setup with prefilled reagents and disposables
Eliminate contamination
with prefilled reagents and disposables, an integrated HEPA filter, and synchronized
stage movement
...
Track sample identification
with the supplied bar-code scanner
...
Navigate easily
with the intuitive software and touch-screen monitor
...
Using this technology, the MagNA Pure LC Instrument and the MagNA
Pure Compact Instrument perform all steps of the procedure (sample uptake, lysis, binding to magnetic particles, wash steps, and elution) in specially designed pipette tips
(Figure 59)
...
Furthermore, the application of the magnetic bead technology within the MagNA Pure LC Instrument and the MagNA Pure Compact Instrument
eliminates the need for vacuum pumps or tubing and the risk of cross contamination
...
7
224
To learn more about MagNA Pure LC and the MagNA Pure Compact Instruments, visit
www
...
com
Nucleic Acid Isolation and Purification Manual
Premium Performance Products
for PCR and RT-PCR
Introduction
PCR Product Selection Guide
RT-PCR Product Selection Guide
PCR Enzyme dNTPacks
Master Mixes
Real-Time PCR Instruments
Reagents for Real-Time PCR
226
227
228
229
230
231
232
8
Introduction
Introduction
The key to successful PCR is the preparation of high-purity nucleic acid templates
...
In particular, clinical
samples like blood and body fluids may contain inhibitory factors such as EDTA,
heparin and porphyrins (hemoglobin)
...
g
...
Although all of our products will perform in a broad array of applications,
you can leverage products’ individual characteristics for your specific applications
...
These products have been function tested to perform optimally with the
nucleic acid purification products listed in this manual
...
roche-applied-science
...
html)
Biochemicals Catalog
iochemica newsletters, (also available on our web site:
B
www
...
com)
Brochure “Tools for Amplification”
...
With more than 50 years of enzyme-purification expertise, Roche Applied
Science has mastered the science of enzyme blending to bring you premium performance PCR and RT-PCR products
...
Insist on excellent fidelity
Robust buffer systems facilitate outstanding yields and accuracy without optimization
...
Amplify longer fragments
More full-length product – up to 35 kb – is obtained with higher fidelity and yields
from complex genomic DNA
...
Simultaneously reverse transcribe rare and abundant RNA – without distorting gene
expression levels
...
Transcriptor First Strand cDNA Synthesis Kit
everse transcribe rare and abundant RNA without altering gene expression levels
...
enefit from a kit that contains all reaction components, specifically designed for
B
use with real-time PCR instruments
...
erform full-length cDNA synthesis in only 10 minutes
...
D
inimize pipetting steps and prevent contamination
...
Product Selection Guide
A comprehensive portfolio for cDNA synthesis and RT-PCR
Two-Step RT-PCR
8
228
Product Size
Yield
Sensitivity
Difficult Templates
Reaction Temperature
Full-Length cDNA
Performance in qRT-PCR
Proofreading Activity
One-Step RT-PCR
up to 14 kb
up to 14 kb
up to 14 kb
����
����
����
up to 6 kb
���
���
���
���
����
����
����
����
��
42 — 65°C
45 — 55°C
42 — 65°C
42 — 65°C
����
����
����
���
����
����
����
no
yes
no
—
no
Transcriptor Reverse
Transcriptase
Transcriptor High Fidelity
cDNA Synthesis Kit
Transcriptor First Strand
cDNA Synthesis Kit
Transcriptor One-Step
RT-PCR Kit
Nucleic Acid Isolation and Purification Manual
Master Mixes
When accuracy …
… meets fidelity
6
...
0 x 104
Accuracy
4
...
0 x 104
2
...
0 x 104
0
Transcriptor High Fidelity
Reverse Transcriptase
Commonly used M-MuLV
Reverse Transcriptase
Figure 60: Accuracy of Transcriptor High Fidelity Reverse
Transcriptase and a commonly used M-MuLV Reverse
Transcriptase
...
The error rate of the Transcriptor High
Fidelity Reverse Transcriptase is a mean value of four independent
experiments in which at least 3
...
For
the M-MuLV reverse transcriptase, 4
...
The accuracy is represented as error rate-1
...
Total RNA (1 μg from human muscle total RNA for the 2
...
3 kb and 9
...
4 kb fragment)
was reverse transcribed using different reverse transcriptases,
according to the manufacturers’ recommendations
...
PCR Enzyme dNTPacks
Choose Roche Applied Science’s dNTPacks, convenient products that combine PCRGrade Nucleotides, thermostable enzymes and enzyme blends, and all associated components such as buffers and PCR-enhancing additives
...
Each dNTPack contains the additive-free sodium salt nucleotides as a ready-to-use mix
(10 mM of each dNTP)
...
afeguard your precious reaction components
S
The extensive investment in generating template material should not be risked by
using nucleotides from another supplier
...
enefit from an attractive price
B
Thermostable DNA polymerases and premixed solutions of PCR-Grade Nucleotides
are provided in one economical package
...
Roche Applied Science’s Master mixes are ready-to-use, double-concentrated solutions
that contain all the reagents (except PCR primers and template) needed for running
PCR: Polymerase or polymerase blend, magnesium chloride, double-concentrated
reaction buffer, and nucleotides (dATP, dCTP, dGTP, dTTP, 0
...
implify PCR setup
S
Only two pipetting steps are needed before starting PCR
...
njoy convenient packaging options
E
Master mixes can be obtained in 50 ml quantities designed for high throughput
applications, or in multiple vials containing smaller volumes
...
nsist on room temperature stability
I
Roche Applied Science’s master mixes are compatible with robotics pipetting stations
because of tested stability at room temperature
...
This type of analysis allows the amplification and fluorescent detection steps to
be performed by a single instrument in a single tube with data recorded online
...
Because PCR itself and the detection of PCR products
occur in the same reaction (vessel), this set-up is also called “homogeneous PCR”
...
It includes instrumentation, software, reagents, technical support, and
application-specific kits
...
0 Instrument,
Cat
...
: 03 531 414 201)
...
0 Instrument is optimized for two fluorescence detection formats:
SYBR Green I and HybProbe probes
...
The LightCycler® 480 System (Cat
...
04 640 268 001 96 well;
Cat
...
04 545 885 001 384 well) is a modular online PCR device
for qualitative or quantitative detection of nucleic acids, mutation
screening and genotyping
...
Offering the sensitivity and accuracy one has come to expect only
from Roche Applied Science´s LightCycler® Carousel-Based System,
the LightCycler® 480 Real-time PCR System goes one step further
providing enhanced 96- or 384-multiwell throughput
...
g
...
For even higher throughputs (up to 1536 reactions in one run) and smaller reaction
volumes (0,5-2µl), a separate plate-based instrument, the LightCycler® 1536 System, is
available
...
lightcycler
...
lightcycler480
...
lightcycler1536
...
For
reagents developed for the LightCycler® Instrument please refer to our website
www
...
com
...
For
details please refer to: www
...
com
...
Revolutionize the way you design and perform real-time qPCR
assays on various real-time PCR instruments
...
For more information, please visit www
...
com/qpcr
...
Tips for Handling Nucleic Acids
Tips for handling nucleic acids
Success of most procedures in molecular biology depends upon the purity and integrity
of nucleic acids
...
These tips
are not intended to cover these topics in detail, but to identify important areas which
should be considered when performing experimental procedures
...
This
procedure minimizes degradation of crude DNA by limiting the activity of endogenous
nucleases
...
Storage of DNA
Store genomic DNA at +2 to +8°C
...
Plasmid DNA and other small circular DNAs can be stored at +2 to +8°C or at –15 to
–25°C
...
Repeated use of a single
sample may lead to shearing
...
It is better to let it air dry
than to use a vacuum, although vacuum drying can be used with caution
...
To help dissolve the DNA, carefully invert the tubes several times after adding buffer and
or tap the tube gently on the side
...
Minimize vortexing of genomic DNA since this can cause shearing
...
Pipetting genomic DNA through small tip openings causes
shearing or nicking
...
Regular pipette tips pose
no problem for plasmid DNA and other small circular DNAs
...
After putting on gloves, avoid
touching contaminated surfaces and equipment with the gloved hands
...
Equipment and disposable items
Use sterile, disposable plasticware whenever possible
...
Electrophoresis tanks for RNA analysis can be cleaned by
wiping them with a solution of SDS (1%), rinsing with water, then rinsing with absolute
ethanol, and finally soaking them in 3% H2O2 for 10 min
...
234
Nucleic Acid Isolation and Purification Manual
Tips for Handling Nucleic Acids
Glass- and plasticware
Treat glass- and plasticware with RNase inactivating agents
...
Autoclaving glassware alone is not sufficient to eliminate
RNases from your experiments
...
1 M
NaOH/1 mM EDTA (or absolute ethanol with 1% SDS), rinsed with DEPC (diethyl
pyrocarbonate) or DMPC1 treated water and heated to +100°C for 15 min in an autoclave
...
Workspace and working surfaces
Designate a special area for RNA work only
...
Also, wipe benches with 100% ethanol
each time prior to use, in order to rid the area of microorganisms
...
Separate reagents used for
RNA work from ‘general use reagents’ in the laboratory
...
1% DEPC (DMPC) overnight at room temperature and then
autoclaved
...
Alternatively, solutions can be made with DEPC-treated and autoclaved water in RNase-free
glassware
...
Dedicate one bottle of Tris for RNA work only
...
Autoclaving without treatment with DEPC (DMPC) is not sufficient for inactivating
RNases
...
For the best results, use
either fresh samples or samples that have been quickly frozen in liquid nitrogen and
stored at –70°C
...
RNase inhibitors
RNase inhibitors can be used to protect RNA from degradation both during isolation
and purification and also in downstream applications such as reverse transcription into
cDNA by RT-PCR, in vitro RNA transcription/translation reactions and RNA-dependent
in vitro functional assays
...
Protector RNase Inhibitor is fully active over a
broad temperature range of 25 to +55°C
...
This is advantageous when performing reverse transcription reactions at
elevated temperatures to overcome secondary structure in RNA
...
To protect difficult RNA
samples the amount of Protector RNase Inhibitor could be increased up to 16 times the
standard concentration without interfering with the performance of enzymes used in the
assay
...
1
...
235
Conversion Tables and Formulas
Storage of RNA
Store RNA at –70° to –80°C, as aliquots in ethanol or isopropanol
...
Centrifuge the RNA and resuspend in the appropriate
RNase-free buffer before use
...
When working with RNA, place
all samples on ice
...
Dissolve RNA by adding RNase-free buffer or water, then standing the tube on ice for 15
min
...
Temperature sensitivity
Although DNA is relatively stable at elevated temperatures (+100°C), most RNA is not
(except for short RNA probes which are stable for 10 min at +100°C)
...
Instead, to melt out secondary structures, heat RNA to +65°C for 15 min in the presence
of denaturing buffers
...
Conversion Tables and Formulas
Metric prefixes (International System)
Bigger
Smaller
Symbol
Prefix
Multiplication factor
Symbol
Prefix
Multiplication factor
T
tera
1012
m
milli
10–3
G
giga
10
9
µ
micro
10–6
M
mega
106
n
nano
10–9
k
kilo
103
p
pico
10–12
f
femto
10–15
a
atto
10–18
Moles, molar, molarity
9
Term
Symbol
Meaning
Example
Mole
mol
Absolute amount of
a substance
1 mol 6
...
022 x 1023 molecules/liter
a one molar solution a solution
with a molarity of one
Example of molar equivalence
0
...
Example how to calculate: 0
...
236
Nucleic Acid Isolation and Purification Manual
Conversion Tables and Formulas
Physical conversions and formulas
Centrifugal force conversion (Dyson, 1991)
RCF = (1
...
, 1989)
Compound
Molecular weight
(MW)
lmax (pH 7
...
2
259
15,400
dATP
491
...
2
271
9,000
dCTP
467
...
2
253
13,700
dGTP
507
...
2
260
10,000
dTTP
482
...
Spectrophotometric equivalents
Nucleic acid
Molarity
(in nucleotides)
double-stranded DNA
50 µg/ml
0
...
10 mM
single-stranded RNA
40 µg/ml
0
...
06 – 0
...
Calculations
9
Determining purity of nucleic acid preparations
For pure DNA:
A260/A280 $1
...
0
An A260/A280 ratio of <1
...
0 (RNA) means the nucleic acid preparation
contains contaminations (e
...
, protein), or phenol
...
, 1988)
For molecular weight of
Use this calculation
DNA base pair (sodium salt)
1 base pair = 665 daltons
double-stranded DNA
molecule
(number of base pairs) x (665 daltons/base pair)
single-stranded DNA molecule
(number of bases) x (325 daltons/base)
single-stranded RNA molecule
(number of bases) x (340 daltons/base)
oligonucleotide
For dephosphorylated oligonucleotides:
[(number of A x 312
...
2) +
(number of C x 288
...
2)] – 61
For phosphorylated oligonucleotides:
[(number of A x 312
...
2) +
(number of C x 288
...
2)] + 17
Conversions between picomoles and micrograms of DNA
E For double-stranded DNA (dsDNA):
To convert
Calculate*
pmol to µg
660pg
1µg
pmol N
6
= µg
1pmol
10 pg
µg to pmol
pmol
106pg
µg
= pmol
1µg
660pg
* N = number of base pairs in DNA; 660, average molecular weight of a base pair
...
1 x 10
13
0
...
52
9
...
66
pUC18/19 DNA
2686 bp
0
...
4 x 1011
1
...
35
2
...
88
M13mp18/19 DNA
7250 bp
0
...
3 x 10
11
4
...
03
1
...
01
Protein data
Conversion between DNA and protein
1 kb of DNA = 333 amino acids of coding capacity = a protein of 3
...
Buffers and Gels for Electrophoresis
Running buffers for non-denaturing gel electrophoresis
(Adapted from Brown, 1991)
Buffer
Components (1 x buffer)
Recipe for 1 liter of 10 x buffer
TBE
(Tris-borate)
89 mM Tris-borate (pH 8
...
0 g Tris-base
55
...
3 g Na2EDTA
TAE
(Tris-acetate)
40 mM Tris-acetate (pH 7
...
4 Tris-base
11
...
0 ml 0
...
0)
TPE
(Tris-phosphate)
89 mM Tris-phosphate
2 mM Na2EDTA
108
...
5 ml phosphoric acid (85%)
40
...
5 M Na2EDTA (pH 8
...
5
1 x TBE is the standard running buffer for DNA separation in polyacrylamide gels
...
Store at room temperature and discard any solution that
develops a precipitate
...
40
2000 – 30,000
0
...
00
1000 – 15,000
500 – 10,000
1
...
50
200 – 4000
2
...
25% (w/v) Bromphenol blue
0
...
25% (w/v) Bromphenol blue
0
...
25% (w/v) Bromphenol blue
0
...
, 1996)
DNA, RNA
Alkaline gels
(McDonell et al
...
0
(neutral pH, because
glyoxal dissociates from
nucleic acids at pH
>8
...
)
Same as gel buffer
(The running buffer
must be circulated
during electrophoresis
...
025% (w/v) Bromocresol green
0
...
0
Incubate at +50°C for 1 h;
cool, then add:
2 µl 10 mM sodium
phosphate, pH 7
...
4% (w/v) Bromphenol
blue
Prepare freshly!!
250 µl formamide
(deionized)
83 µl formaldehyde,
37% (w/v)
50 µl 10 x MOPSbuffer*
0
...
* 10 x MOPS:
00 mM Morpholinopropanesulfonic acid, pH 7
...
, 1989)
% Acrylamide
DNA size range (bp)
Bromphenol Blue*
Xylene Cyanol*
3
...
0
75 – 500
65
260
8
...
0
35 – 250
20
70
15
...
0
* Approximate size of DNA fragments (in base pairs) with which the dyes would migrate
...
, 1989)
% Acrylamide
Bromphenol Blue*
Xylene Cyanol*
5
...
0
26
106
8
...
0
12
55
* Approximate size of DNA fragments (in nucleotides) with which the dyes would migrate
...
)
Agaroses and molecular weight markers
see inside back cover page
...
8
DNA, RNA stain in gel
electrophoresis
1 – 10 µg/ml in staining
solution
Bromphenol Blue
670
...
01 – 0
...
3
Fluorescent dye for DNA/
RNA in density gradients
and electrophoresis gels
1 µg/ml in running
buffer or staining solution
Xylene Cyanol
554
...
01 – 0
...
Other Useful Information
Length of rRNAs from various species
(Lewin, 1987)
Species
242
Length (bases)
E
...
cerevisiae)
18 S rRNA
28 S rRNA
2000
3750
Drosophila
(D
...
, Masseyeff et al
...
Serum, unlike plasma,
does not contain fibrinogen; serum also lacks several clotting factors present in plasma
...
It is comprised of leukocytes and thrombocytes
...
M
...
(1988) (Eds), Current Protocols in Molecular Biology, John Wiley and
Sons, New York
Bailey, J
...
and Davidson, N
...
Biochem
...
K
...
L
...
Virol
...
A
...
Bios Scientific Publishers, Academic Press
...
and Davidson, N
...
4, 1539
Czihak, G
...
and Ziegler, H
...
J
...
(T
...
2, Oxford University Press, Oxford, UK
Farrel, R
...
(1983) RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Academic Press, New York
Lewin, B
...
148
Masseyeff, R
...
, Albert, W
...
and Staines, N
...
, Methods of Immunological Analysis, Cells
and Tissues, VCH
McDonell, M
...
, Simon, M
...
and Studier, F
...
(1977) J
...
Biol 110, 119
Rueger, B
...
, Obermaier, I
...
(1996) Biochemica 3,
35 − 38
Sambrook, J
...
F
...
(1989) Molecular Cloning, A Laboratory
Manual, 2nd edition, Cold Spring Harbor Laboratory Press
9
Wallace, R
...
et al
...
6, 3543
Appendix
243
Notes
9
244
Nucleic Acid Isolation and Purification Manual
Ordering Guide
Isolation and Purification of DNA
Isolation and Purification of RNA
MagNA Lyser Instrument and Accessories
Automated Isolation using the MagNA Pure LC Instrument
Automated Isolation using the MagNA Pure Compact Instrument
Companion Reagents for Isolating Nucleic Acids
246
247
248
248
249
250
10
Isolation and Purification of DNA
Isolation and Purification of DNA
10
246
Product
Agarose Gel DNA Extraction Kit*
for the elution of DNA from agarose gel slices
DNA Isolation Kit for Cells and Tissue*
for the extraction of genomic DNA from cells and tissues ranging in size
from 50 to 150 kb
DNA Isolation Kit for Mammalian Blood*
for the isolation of intact genomic DNA from mammalian whole blood,
lymphocyte, or buffy coat samples
High Pure 96 UF Cleanup Kit*
for high-throughput purification of PCR products by ultrafiltration
High Pure 96 UF Cleanup Plates*
for high-throughput purification of PCR products by ultrafiltration
High Pure PCR Cleanup Micro Kit*
for purification of products from PCR and other reactions
High Pure PCR Product Purification Kit*
for the purification of PCR reaction products
High Pure PCR Template Preparation Kit+
for isolating genomic nucleic acids for PCR, restriction enzymes
analysis, and Southern blotting
High Pure Plasmid Isolation Kit*
small scale “mini-preps” for sequencing, PCR, and cloning
High Pure Viral Nucleic Acid Kit+
for isolating viral DNA and RNA for PCR or
RT-PCR
High Pure Viral Nucleic Acid Kit Large Volume+
for the isolation of viral nucleic acids for PCR and RT-PCR
High Pure Viral Nucleic Acid Buffer Set+
for the isolation of viral nucleic acids for PCR and RT-PCR
Quick Spin Columns for radiolabeled DNA*
Sephadex G-25
Quick Spin Columns for radiolabeled DNA*
Sephadex G-50
mini Quick Spin DNA Columns*
mini Quick Spin Oligo Columns*
Genopure Plasmid Midi Kit*
for medium-scale (midi) preparation of plasmid DNA
Genopure Plasmid Maxi Kit*
for large-scale (maxi) preparation of plasmid DNA
Genopure Buffer Set for Low-Copy Number Plasmids*
for isolation of low-copy number plasmid DNA in combination with the
Genopure Plasmid Kits
Red Blood Cell Lysis Buffer
for the preferential lysis of erythrocytes in human whole blood
TriPure Isolation Reagent*
for the simultaneous isolation of DNA, RNA, and denatured proteins
from cells or tissues of human, plant, yeast, bacterial, or viral origin
...
No
...
Not for use in diagnostic procedures
...
Nucleic Acid Isolation and Purification Manual
Isolation and Purification of RNA
Isolation and Purification of RNA
Product
High Pure FFPE RNA Micro Kit+
for isolation of total RNA from formalin-fixed, paraffin-embedded tissue
High Pure RNA Isolation Kit*
for small scale preparations of total RNA (free of genomic DNA) from
blood, cultured cells, yeast, and bacteria
High Pure RNA Tissue Kit*
for the isolation of total RNA from tissue
High Pure RNA Paraffin Kit*
for the isolation of total RNA from fresh-frozen and formalin-fixed,
paraffin-embedded tissues
High Pure Viral RNA Kit+
for the isolation of viral RNA for RT-PCR
High Pure miRNA Isolation Kit*
for purification of small or total RNA from cells, tissue, FFPE tissue
sections
mRNA Capture Kit*
for the immobilization of poly(A+) RNA in a PCR tube, prior to reverse
transcription
mRNA Isolation Kit*
for the affinity isolation of poly(A+) RNA
mRNA Isolation Kit for Blood/Bone Marrow*
for the isolation of mRNA from whole blood or bone marrow lysates,
preserved with the RNA/DNA Stabilization Reagent for Blood/Bone
Marrow
RNA/DNA Stabilization Reagent for Blood/Bone Marrow*
for instantaneous stabilization of DNA and RNA in blood or bone
marrow samples prior to isolation mRNA or DNA (used with the mRNA
Isolation Kit for Blood/Bone Marrow)
Quick Spin Columns for radiolabeled RNA*
Sephadex G-25
Quick Spin Columns for radiolabeled RNA*
Sephadex G-50
mini Quick Spin RNA Columns*
Red Blood Cell Lysis Buffer
for the preferential lysis of erythrocytes in human whole blood
Streptavidin Magnetic Particles
TriPure Isolation Reagent*
for the simultaneous isolation of DNA, RNA, and denatured proteins
from cells or tissues of human, plant, yeast, bacterial, or viral origin
...
No
...
Not for use in diagnostic procedures
...
10
Ordering Guide
247
Automated Isolation
MagNA Lyser Instrument and Accessories
Product
MagNA Lyser
Instrument#
MagNA Lyser
Rotor#
MagNA Lyser
Rotor Cooling
Block#
MagNA Lyser
Green Beads#
Application
Automated homogenization of tissue samples
Cat
...
03 358 968 001
03 358 976 001
Holds up to 16 sample tubes for tissue homogenization 03 359 093 001
Pack Size
1 instrument
plus accessories
1 rotor
Houses the MagNA Lyser Rotor to maintain the
temperature of the samples at +2 to +8°C
03 359 085 001
1 cooling block
Specially designed ceramic beads to achieve optimal
homogenization of various sample materials
03 358 941 001
100 tubes
(prefilled with
ceramic beads)
Automated Isolation using the MagNA Pure LC Instrument
Product
MagNA Pure LC
Instrument+
10
Application
Robotic workstation for fully automated nucleic acid
preparation and filling of LightCycler® Capillaries,
96‑well PCR plates, and tubes suitable for the most
commonly used PCR instruments
Reagent Kits for Isolation of genomic DNA
Ready-to-use reagents for the isolation of high-quality
MagNA Pure LC
genomic DNA from whole blood, white blood cells,
DNA Isolation
+
peripheral blood lymphocytes, and cultured cells, using
Kit I
the MagNA Pure LC Instrument
...
Ready-to-use reagents for the isolation of high-quality,
MagNA Pure LC
intact bacterial or fungal DNA from the most difficultDNA Isolation
Kit III+ (Bacteria & to-process research sample materials, using the
MagNA Pure LC Instrument
...
Reagent Kits for Isolation of viral and total Nucleic Acids
Ready-to-use reagents for the purification of viral
MagNA Pure LC
Total NA Isolation nucleic acid from serum, plasma and whole blood,
using the MagNA Pure LC Instrument
Kit+
Ready-to-use reagents for the purification of viral
MagNA Pure LC
Total NA Isolation nucleic acids from large amounts (up 1 ml) of serum
and plasma, using the MagNA Pure LC Instrument
Kit+ – Large
Volume
Reagent Kits for Isolation of total RNA
Ready-to-use reagents, developed to maximize yield of
MagNA Pure LC
RNA Isolation Kit+- purified total RNA with superior quality isolated from
High Performance blood, blood cells or culture cells, using the MagNA
Pure LC Instrument
#
+
248
Cat
...
12 236 931 001
Pack Size
1 instrument
plus accessories
03 003 990 001
1 kit
(192 reactions)
03 186 229 001
1 kit
(192 reactions)
03 264 785 001
1 kit
(192 reactions)
03 310 515 001
1 kit
(96 – 288
reactions)
03 038 505 001
1 kit
(192 reactions)
03 264 793 001
1 kit
(192 reactions)
03 542 394 001
1 kit
(192 reactions)
CE marked /for USA for laboratory use
For general laboratory use
...
g
...
Kit II+ (Tissue)
Ready-to-use reagents for the purification of mRNA
MagNA Pure LC
from up to 1 x 107 WBCs (white blood cells) or PBMCs
mRNA HS Kit
(peripheral blood mononuclear cells), using the
MagNA Pure LC Instrument
Cat
...
03 330 591 001
Pack Size
1 kit
(192 reactions)
03 004 015 001
1 kit
(192 reactions)
03 172 627 001
1 kit
(192 reactions)
03 267 393 001
1 kit
(192 reactions)
Automated Isolation using the MagNA Pure Compact Instrument
Product
MagNA Pure
Compact
Instrument+
Application
Cat
...
Automated nucleic acid isolation for a broad range of 03 731 146 001
applications
Kits and Reagents for the Isolation of DNA
E enomic DNA from mammalian whole blood or
G
MagNA Pure
cultured cells
Compact Nucleic
Acid Isolation
E iral nucleic acids from plasma or serum
V
Kit I+
E ample volume range 100 μl – 400 μl
S
E enomic DNA from mammalian whole blood or
G
MagNA Pure
cultured cells
Compact Nucleic
Acid Isolation
E iral nucleic acids from plasma or serum
V
Kit I+ - Large
E ample volume range 500 μl – 1000 μl
S
Volume
E NA from bacteria in many different sample
D
MagNA Pure
types, such as urine, BAL (bronchoalveolar
Bacteria Lysis
lavage), sputum, CSF, swabs, or bacterial cultures
Buffer+
G
MagNA Pure DNA E enomic DNA from mammalian tissue
...
10
For detailed information about the MagNA Pure LC System and accessories, please visit
www
...
com
Ordering Guide
249
Companion Reagents for Isolating Nucleic Acids
Companion Reagents for Isolating Nucleic Acids
Product
Proteases
Proteinase K*
recombinant PCR Grade, solution
Description
Application
Non-specific enzyme for digesting
cellular proteins
Rapid inactivation of endogenous
nucleases and isolation of nucleic acid
from tissues or cell lines
Proteinase K*
recombinant PCR Grade, lyophilizate
See above
See above
Pronase*
from Streptomyces griseus
Pronase is a various mixture of
proteases with different proteolytic
activities
In conjunction with other enzymes (e
...
collagenase, trypsin), pronase is
suitable for the isolation of a variety of
cell types
Endonuclease for double- and singlestranded DNA, free of ribonuclease
und protease
RNase mixture, free of contaminating
DNases
Pyrimidine-specific endonuclease that
acts on single-stranded RNA
Endonuclease that cleaves RNA in
RNA:DNA hybrids
Isolation of DNA-free RNA
Recombinant from rat lung
Protects mRNA and total RNA during
isolation and cDNA synthesis
Nucleases and nuclease inhibitors
DNase I recombinant, RNase-free*
from bovine pancreas, expressed in
Pichia pastoris
RNase, DNase-free*
RNase A*
RNase H*
Protector RNase Inhibitor*
Crude mixture of RNases
...
2 – 15 kbp
Electrophoretic separation of nucleic
acids, of PCR products genotyping
allele sizing, STR analysis
Electrophoretic separation of nucleic
acids, especially high molecular weight
DNA
Separation and purification of nucleic
acids by density
Carrier for the precipitation of nucleic
acids
Protein denaturing agent; component
of buffers
Component of buffers; stabilizes
enzymes
RNA carrier for precipitation
* For life science research only
...
250
Nucleic Acid Isolation and Purification Manual
Companion Reagents for Isolating Nucleic Acids
Features
Cat
...
Ideal for isolation of nucleic acids, since it is virtually free of RNases and
DNases
Enhances isolation of intact nucleic acids, since it rapidly inactivates
e
ndogenous nucleases
Especially suited for isolation of PCR templates, since preparation has
m
inimized level of contaminants
...
25 ml
5 ml
25 ml
03 115 836 001
03 115 879 001
03 115 801 001
03 115 852 001
25 mg
100 mg
2 x 250 mg
4 x 250 mg
otal degradation of proteins during the isolation of DNA and RNA (it is not
T
necessary to let pronase self-digest prior to use)
10 165 921 001
11 459 643 001
1 g (non-sterile)
5 g (non-sterile)
deal for the isolation of RNA, since it is virtually free of RNases
I
ely on a recombinant animal-free enzyme
R
04 716 728 001
10,000 U
Ideal for isolation of DNA, since it is virtually free of DNases
Unlike most RNase preparations, does not need to be boiled prior to use
Used in many DNA isolation procedures
11 119 915 001
500 µg
10 109 142 001
10 109 169 001
10 786 349 001
10 786 357 001
25 mg
100 mg
25 units
100 units
ctive over a broad pH range (pH 5–8)
A
irtually free of endonucleases and DNA nicking activities
V
ay be used in one-step or two-step RT-PCR
M
sed in many DNA isolation procedures
U
03 335 399 001
03 335 402 001
2000 units
10000 units
10 109 134 001
500 mg
Can be used for the analysis of PCR product, examination of restriction enzyme
digest of plasmid, cosmid and l-phage DNA and electrophoresis of RNA
Resolve PCR fragments (50 bp – 1 500 bp)
Virtually free of DNases and RNases
Discriminate between fragments that differ in only 4 bp of length
uitable for analytical and preparative isolation
S
irtually free of DNases and RNases
V
eparation of high molecular weight DNA (PFGE)
S
Very highly purified preparation
Prepared especially for molecular biology procedures
Unlike tRNA, will not interfere with downstream procedures
11 685 660 001
11 685 678 001
11 816 586 001
11 816 594 001
100 g
500 g
100 g
500 g
11 388 983 001
11 388 991 001
100 g
500 g
10 757 306 001
1 kg
10 901 393 001
20 mg
ery low absorbance at 260 and 280 nm, so it doesn’t interfere with UV
V
detection of nucleic acids
tronger protein denaturant than guanidine hydrochloride
S
ighly purified
H
irtually free of enzyme inhibitors
V
Readily soluble salt
11 685 929 001
500 g
11 332 473 001
5 x 10 ml
10 108 626 001
100 mg
liminates potential source of PCR inhibition
E
Ordering Guide
Pack Size
10
251
Companion Reagents for Isolating Nucleic Acids
Companion Reagents for Isolating Nucleic Acids, continued
Product
Description
Additional reagents required in some purification procedures
Anionic detergent; highly purified
Sodium Dodecyl Sulfate (SDS),
electrophoresis grade*
Tris*
Biological buffer, crystalline
Triton X-100*
Non-ionic detergent
Application
Denaturation of proteins before or
during electrophoresis; dissociation of
protein-nucleic acid complexes
Preparation of buffers, pH 7–9, for
biological and biochemical applications
Solubilization agent for proteins
10
252
Nucleic Acid Isolation and Purification Manual
Companion Reagents for Isolating Nucleic Acids
Features
Cat
...
Pack Size
Rapidly and completely denatures most proteins
Particularly suited for gel electrophoresis
11 667 289 001
1 kg
Virtually free of proteases, RNases, and DNases
pK 8
...
SYBR is a registered trademark of Molecular Probes, Inc
...
RNAlater is a registered trademark of Ambion, Inc
...
Disclaimer
* For life science research only
...
+
For general laboratory use
...
18
Affinity purification, overview of
...
240
Agarose Gel DNA Extraction Kit
...
see inside back cover page
...
225
Automated Nucleic Acid Isolation
...
181
Blood
–– dried spots
...
23
–– mouse, DNA from
...
23
C
Carrier RNA
...
95, 100, 105
Cell disruption
...
14
Centrifugation, overview of
...
250
Concentrated DNA, preparing, see
–– Agarose Gel DNA Extraction Kit
...
95, 100, 105
D
11
256
Deparaffinization procedure
–– for FFPE tissue in reaction tube
...
72
Differential Display RT-PCR, preparing RNA for, see
–– High Pure RNA Isolation Kit
...
122
–– hints for handling
...
234
–– simultaneous isolation of RNA and protein
...
234
DNA isolation from
–– agarose gel slices
...
23, 27
–– blood, buffy coat, cultured cells
...
25
–– formalin-fixed, paraffin-embedded tissue sections
...
30
–– guinea pig blood
...
156
–– mammalian tissue
...
156
–– mouse tail
...
28
–– PCR product mix
...
143
midi prep
...
49
–– rat blood
...
23
DNA Isolation Kit for Cells and Tissues
...
154
DNase treatment
...
15
Erythrocytes, in blood
...
14
Expand Amplifciation, preparing DNA for, see
–– High Pure PCR Template Preparation Kit
...
14
F
Fecal cultures, preparing DNA from, see
–– High Pure PCR Product Purification Kit
...
21
Formalin-fixed tissue sections, preparing DNA from, see
–– High Pure FFPE RNA Micro Kit
...
21
Formulas, reference
...
208
Gel, see Agarose gel
...
236
Genomic DNA
...
150
Genopure Plasmid Maxi Kit
...
136
Glass fibre fleece
...
235
Guinea pig blood, preparing DNA from
...
127
High Pure Extender Assembly
...
69
High Pure miRNA Isolation Kit
...
34
High Pure PCR Product Purification Kit
...
21
High Pure Plasmid Isolation Kit
...
55
High Pure RNA Paraffin Kit
...
64
High Pure Viral Nucleic Acid Kit
...
105
Index
11
257
High Pure Viral RNA Kit
...
127
Hints for handling nucleic acids
...
14
Ion exchange chromatography, overview of
...
14
L
LightCycler® 480 Instrument
...
231
M
MagNA Lyser Instrument
...
223
MagNA Pure LC Instrument
...
198
Magnetic particle separator
...
230
Mechanical disruption of cells
...
198
mini Quick spin Columns
–– DNA
...
214
–– RNA
...
90
–– tissue
...
156
–– tail, preparing DNA from
...
184
–– Isolation Kit
...
198
N
Nucleic acids
–– hints for handling
...
14
O
Ordering information
...
10
P
11
258
Paraffin-embedded tissue sections, preparing DNA from, see
–– High Pure PCR Template Preparation Kit
...
75
preparing RNA from, see High Pure RNA Paraffin Kit
PCR Enzyme dNTPacks
...
227
Poly(A)+ RNA, preparing
...
234
–– for handling RNA
...
250
Protein, simultaneous isolation with DNA and RNA
...
38
Purification of PCR products from solution
...
14
Q
Quantitation of
–– DNA
...
237
Quick Spin columns
–– for purifying radiolabeled DNA
...
210
R
RACE, preparing RNA for, see
–– High Pure RNA Isolation Kit
...
210
Rat blood, preparing DNA from
...
231
Red Blood Cell Lysis Buffer, see
–– High Pure RNA Isolation Kit
...
170
Ribonuclease, see RNase digestion
RNA
–– hints for handling
...
173
–– temperature sensitivity of
...
197
RNA isolation from
–– bacteria
...
88
–– formalin-fixed, paraffin-embedded tissue sections
...
59
–– mouse tissues
...
80
–– yeast
...
24
S
Sections, fixed tissue, preparing DNA from, see
–– High Pure PCR Template Preparation Kit
...
75
preparing RNA from, see High Pure RNA Paraffin Kit
Selection Guide
...
42
Silica Adsorption, overview
...
122
Single-stranded oligonucleotides, purifying, see
–– Agarose Gel DNA Extraction Kit
...
152
Index
11
259
Stabilization
–– of blood or bone marrow lysates
...
235
Streptavidin-coated
–– magnetic particles
...
184
T
Tables and formulas, conversion
...
21
–– High Pure RNA Paraffin Kit
...
170
Troubleshooting Procedures for High Pure Kits
...
95
V
Vacuum Manifold, see
–– High Pure 96 UF Cleanup Kit
...
1 kbp - 30 kbp
(Standard size range)
–564
–564
–831
-947
–1375
–1584
–2027
–1904
–3530
–5148, 4973
–4268
–7743
–74
–421
–697
–925
–1150
-1489
–1882
–2322
–2690
–3140
–4254
–5526
–154
–234, 220
–298
–517
–453
-394
Agarose LE
(0
...
15-2
...
05 kbp - 1
...
08-8
...
019-1
...
072-1
...
12-23
...
12-21
...
075-12
...
07-19
...
All rights reserved
...
roche-applied-science