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DNA extraction: Comparison of
methodologies
Ambika B Gaikwad
ambika@nbpgr
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in

Principle
Good quality DNA is a prerequisite for all experiments of DNA manipulation
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This is brought about by
disruption of the tissue in a mortar and pestle aided by liquid nitrogen and the various
components of the homogenization or extraction buffer followed the precipitating and
purification methods employed
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, the
tissue type along with the concentration of DNA finally required determine the methodology
of DNA extraction to be followed by the experimenter
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1983 and Saghai Maroof et al
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In addition to these basic
protocols, a panorama of DNA isolation kits
based on either anion exchange
chromatography or silica gel membranes are available commercially
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The extraction buffer
This includes a detergent such as cetyl trimethyl ammonium bromide(CTAB) or SDS
which disrupts the membranes, a reducing agent such as B mercaptoethanol which
helps in denaturing proteins by breaking the disulfide bonds between the cysteine
residues and for removing the tanins and polyphenols present in the crude extract, a
chelating agent such as EDTA which chelates the magnesium ions required for
DNase activity , a buffer which is almost always Tris at pH 8 and a salt such as
sodium chloride which aids in precipitation by neutralizing the negative charges on
the DNA so that the molecules can come together
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Phenol chloroform extraction
Nucleic acid solutions commonly contain undesirable contaminants that are chiefly
made of proteins
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0); a volume of phenol: chloroform: isoamyl alcohol (25: 24:
1

1) and chloroform: isoamyl alcohol ( 24:1)
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The contaminants are denatured and and accumulate in the
organic phase or in the marginal layer between the two phases and the nucleic acids
are preserved in the aqueous phase
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C
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This requires diluting the nucleic acid with a monovalent salt , adding
alcohol to it and mixing gently
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The salts and alcohol remnants are removed by
washing with 70% alcohol
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2(final volume 0
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2M), ammonium
acetate (2- 2
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8M) and potassium chloride
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D
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Purification of DNA
The DNA is purified by incubating the nucleic acid solution with RNase A (10mg/ml)
at 37°C and reprecipitation following phenol: chloroform extraction to remove the
RNase
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Reagents
1
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4 M Na Cl
100 mM Tris (pH 8
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0)
2% -Mercaptoethanol
2% CTAB
2
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Saturated phenol pH 8
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Chloroform : isoamylalcohol ( 24:1) mixture
5
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0
10 mM Tris
6
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5, 15 mM NaCl
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Cool to
room temperature
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7
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Transfer to 50 ml centrifuge tube with 10 ml extraction buffer maintained at 65°C in a
water bath
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Incubate at 65°C for one hour
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Allow to come to room temperature
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Mix gently by makinf a figure of ‘8”
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Transfer aqueous phase to a fresh centrifuge tube
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6 volume of chilled
isopropanol and let the DNA to precipitate for 30 min
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Spool out the DNA
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Add 0
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Mix gently and keep it at room temperature for 15 min
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Decant off and dry the pellet under
vacuum or air dry
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Add RNAse A (10 l) and incubate at 37°C for one hour
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Extract twice with
chloroform : isoamyl alcohol
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0 times of the total volume
chilled ethanol
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Remove extra salts by two washings with
70% ethanol
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Add minimum volume of TE (10:1)
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Store
at – 20°C
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In chloroform : isoamyl alcohol extraction, the aqueous phase should be
carefully removed and organic phase re-extracted to ensure full recovery of
DNA
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Care should be taken to do the operations as gently as possible
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should be avoided to prevent the shearing of DNA
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All the glassware, plastic ware, pestles and mortars etc
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Care should be taken to prevent cross-contamination
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3

DNA Isolation using kits
These include isolation of nucleic acids using anion-exchange chromatography or silica gel
membrane technology
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Isolation of nucleic acids using anion-exchange chromatography
Nucleic acids are highly charged, linear poly-anions and can therefore be separated from
other components by anion-exchange chromatography
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1 m to 1
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The
large pore size, together with high density of anion-exchange groups, provides a broad
separation range that allows selective separation of nucleic acids from proteins,
polysaccharides and metabolites
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2
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4
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6
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8
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5, 2% CTAB, 1
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4-1 g of plant tissue
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The procedures have the following main steps:
 Preparation of the lysate
 Selective binding of the desired nucleic acid to QIAGEN-tip under low salt conditions
 Washing of the tip with buffers of the moderate salt concentration to remove impurities
 Elution of the nucleic acid with a high salt buffer
 Isopropanol precipitation for desalting
Isolation of nucleic acids using silica-gel based membranes
The DNeasy membrane from QIAGEN combines the binding properties of a silica-gel based
membrane with microspin technology
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Buffer conditions in DNeasy plant procedure are designed to allow
specific adsorption of DNA to the silica-gel membrane and optimal removal of
carbohydrates, polyphenolics and other plant metabolites
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 The cleared lysate is transferred to a new tube and binding buffer and ethanol are added
to promote binding of DNA to the DNeasy membrane
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 Pure DNA is eluted in a small volume of low salt buffer or water
The detailed protocols of QIAGEN Anion-exchange chromatography and silica-gel membrane
technology are available along with their respective kits
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The system provides shearing tubes for simple and fast homogenization as well as filtration
of tissues
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Material
1
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3
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5
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7
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9
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11
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13
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0)
PX1 Buffer
PX2 Buffer
PX3 Buffer
WS Buffer
Rnase A
Plant Genomic DNA Mini Column
Collection Tube
Shearing Tube (For Mini column)

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Procedure

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










Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and quickly
transfer to a sterile 1
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Add 400 l PX1 Buffer and 4 l RNase A stock solution (100 mg/ml) to the tissue
powder and vortex vigorously
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Invert mix
every 2 minutes during incubation
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0), 10 mM Tris-HCl (pH 9
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Add 130 l PX2 Buffer to the lysate, and vortex the mixture
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Place a Shearing tube onto a Collection tube
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Transfer the flow-through lysate from
the Collection tube to a new sterile tube
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Add 0
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Add 1 volume of 98-100% ethanol to the mixture and mix by
pipetting
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g
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Place a DNA easy Plant Mini column onto a Collection tube
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Discard the flow-through
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Transfer the column onto a new 1
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Discard the collection tube and flowthrough
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0), or TE buffer preheated at 65C
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Store eluted DNA at 4C for frequent use or -20°C for long-term storage
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Dellaporta, S
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, Wood, J
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B
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A plant DNA mini preparation: Version II
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2
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A
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M
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A
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W
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Ribosomal
DNA spacer length polymorphism in barley: Mendelian inheritance, chromosomal
location and population dynamics
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Natl
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Sci
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6



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