Notesale: Turn your study into money

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1
...
Know why we perform PCR
1
...
Easier to analyze bigger sample
b
...
Positive control
1
...
If this yields results, proves everything was working properly
2
...
Contains no DNA
2
...
Know why dNTPs are used for PCR, and not ddNTPs
1
...
ddNTPs not used because they’re designed to stop extension
1
...
don’t have OH on 3’ C  can’t continue process
d
...
Denaturing: DNA strands separate
2
...
If correct temperature, will only attach at desired space for
amplification
3
...
Exponential increase in DNA
5
...
DNA
2
...
Taq polymerase
4
...

2
...
Know what the difference between agarose vs acrylamide is and when to
use each gel type
1
...
Large range of separation
2
...
Separates big ranges of things, not well defined for small things
2
...
Small range of separation
2
...
Well defined separation of smaller range of things, not for things
with larger ranges
4
...
Covered with thin layer of water/ethanol

6
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Initiated by addition of TEMED and ammonium persulfate
added
2
...
Don’t want oxygen to get in  hinders polymerization
b
...
Current goes from cathode to anode  DNA moved along with it
2
...
DNA has relatively negative charge
1
...
Current flows from positive to negative (anode to cathode)
1
...
Know by what characteristic DNA gets separated through
electrophoresis
1
...
Larger fragments have slower rate of travel
1
...
Smaller fragments have higher rate of movement
4
...
Also conformation
1
...
Linear typically travels faster than supercoiled, but slower than
nicked circles
1
...
Nicked circles: usually slowest
d
...
Can make standard curve to estimate sizes of unknown DNA fragments
2
...
Can compare to how it’s supposed to look
3
...
Know what sequencing allows us to do
1
...
Unknown DNA sample  can sequence to find code
3
...
Compare sample of known and unknown DNA
b
...
Computer program can’t always figure out what base is supposed to be
where
1
...
Ex
...
Clean reads based on which base looks most promising
1
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Need clean reads to create contig/run BLAST

1
...
Know what a contig is
1
...
Edit both sequences
2
...
Know why ddNTPs are used for sequencing
1
...
Each type of base has its own fluorescent color
1
...
Can eventually figure out sequence this way
4
...
Know what ELISA is and what the acronym stands for
1
...
Show concentration of antigens/antibodies in sample
1
...
Describe the three different types of ELISA that you were taught in lab,
and how we applied the theory in the lab
1
...
Find how much antigen is in sample
2
...
Indirect
1
...
HIV test
1
...
HIV antigens coated on plate
3
...
If positive, antibodies will bind
2
...
Has enzyme bound
3
...
Sandwich
1
...
Pregnancy test
1
...
Dipped in pee with sample, travels to strip with mouse antihCG antibody
1
...
Sample will go to second area; mouse anti-hCG antibody
bound to strip
4
...
If pregnant  color will appear
2
...
Rest of antibody keeps traveling upwards to control strip
1
...
Proves test is working properly
c
...
Monoclonal vs
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Monoclonal: only binds to one type of antigen
2
...
SDS-PAGE
a
...
Method of separating proteins based on molecular weights
2
...
Become linear  can be more effectively separated
3
...
When access to DNA difficult, use this to get protein profile
1
...
With bacteria, can compare unknown to known profile
 identify what it is
2
...
Ex
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Know which direction current flows, Proteins flows, and why
1
...
Binds to polypeptides in ratio of 1
...
Overall negative charge
3
...
Current flows from positive to negative electrode (anode to cathode)
1
...
Plasmid Isolation
a
...
Quadrant method: plate divided into four quadrants, new colony goes in
each
b
...
Circular pieces of bacterial DNA
2
...
Inherited independently
3
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Know what recombination is
1
...
Aim is to clone the DNA
d
...
What each band might stand for
1
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Recombined plasmid
2
...
Foreign DNA

4
...
Why they might not accurately state the size of your plasmid
1
...
May have had recombined DNA removed  not sure
3
...
Why you may have multiple bands
1
...
If so, can get cuts at any such site
2
...
Ex
...
Know the special techniques/chemicals needed to lyse the different types
of cells (gram positive, gram negative)
1
...
Thick peptidoglycan (PGN) wall
1
...
To lyse
1
...
Then can use SDS (sodium dodecyl sulfate)
1
...
Gram negative
1
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Has outer cell membrane as well
3
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Lysozyme  no need
f
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Depends what’s on plate
2
...
Antibiotic
1
...
If no insert present, (most) non-recombinant bacteria should die
1
...
If x-gal
1
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Usually cleaves substrate/creates colored product
2
...
Recombinant will be white (as was ours)
4
...
If non-recombinant
1
...
Can cleave substrate/create blue product

3
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Restriction Enzymes
a
...
Cut DNA at specific sites (recognition sites)
a
...
Usually palindromic (same forwards as backwards)
b
...
Same enzyme can remove foreign DNA when ready
c
...
Know what palindromes are and correctly re-create a second strand of a
palindrome, given the first strand’s sequence
a
...
Ex
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Know the difference between blunt and sticky ends
a
...
No overhanging nucleotides
b
...
More difficult to create restriction enzymes
b
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Overhanding nucleotides
b
...
Easier to reconnect  easier to make recombinant plasmids
d
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Overhanging region acts as template for other overhanging regions
a
...
If AAT overhanging on one fragment, can easily find overhanging
TTA sequence  join quickly/easily
e
...
e
...

a
...
Recombinant plasmid should be largest (contains plasmid and foreign
DNA)
b
...
Insert likely smallest
b
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Ex

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