Laboratory: Unit 3: PCR (pages 54-55)

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Laboratory Unit 3 PCR (pages 54-55) Lecture
PCR primer stock preparation In-Class
Writing abstract for AEM 63 2647-53,
1997 (page
68) Hand In abstract (page 68)
Read AEM 76 8117-8125, 2010 Due Next Class
nothing
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Factors that affect PCR Buffer Mg2, pH,
dNTPs, salt, primer template Template quality
quantity Facilitators (acetamide
2-pyrolidone) Inhibitors (humic acid
hemoglobin) Annealing temperature (primer
length G/C content KCl concentration)
Initial temperature hot start vs. cold
start Cycle number
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Factors that affect PCR Primer sequence
interactions with itself, other primer,
undesired templates Polymerase fidelity
processivity Cycler temperature uniformity
ramp time hot top Reaction tubes material
thickness Ability of exclude or destroy
extraneous DNAs
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Uses of PCR Amplify DNA for sequencing
sequence amplicon (Ex. 3) clone sequence (Ex.
4). Add restriction sites to ends of a
gene. Detect small amounts of DNA. Make
specific mutations overlap extension PCR. Make
"random" mutations error-prone PCR. Amplify
unknown sequences flanking known sequence.
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Uses of PCR Associate physical markers with
genes Random Amplification of Polymorphic DNA
(RAPD). cDNA cloning Rapid Amplification of
cDNA Ends (RACE). Quantitative real-time PCR
dye fluoresces when bound to double-stranded DNA.
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Abuses of PCR Use PCR to build a gene fusion
but fail to sequence the clone PCR is error
prone. PCR is extremely sensitive to DNA
contamination. Quantitative measurements are
more accurate using hybridization instead of
PCR. Aerosol resistant pipet tips are NOT
aerosol proof. Commercial dNTPs are contaminated
with human and animal DNA. Other reagents may
be contaminated too.
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Abuses of PCR Inadequate no-template controls.
Use 1 or 2 controls per sample. Failure to
determine sensitivity for each primer set
reaction cocktail. Failure to remove PCR
inhibitors. Dilution works well requires no
knowledge of inhibitor chemistry.
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Abuses of PCR Cross contamination between
samples when processing multiple samples due to
inadequate cleanup of equipment lack of
physical precautions. Use dedicated equipment,
glove boxes, separate rooms for each step.
Use bleach to decontaminate equipment.
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Abuses of PCR UV irradiate reagents to remove
extraneous DNA. Ineffective if dNTPs present. UV
destroys DNA polymerase if dNTPs absent.
Increase efforts to exclude extraneous
DNA. Reduce PCR cycles until contaminating DNAs
not detected in no-template controls.
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Dissolve 20-base primer (MW 6600) in 1 ml
water. Pipet 5 ul into 495 ul water. Absorbance
_at_ 260 nm 0.61 1 OD260 33 ug/ml for
single-stranded DNA. Concentration of undiluted
primer stock? MW 6600 ? 1M stock 6600 g/l
1 uM stock 6600 ug/l 6.6 ug/ml 5 ul into
495 ul water 1/100 dilution OD260 of diluted
stock 0.61 concentrated stock 0.61 OD260 x
100 x 33 ug/ml/ OD260 2013 ug/ml 2013 ug/ml x
1 uM/6.6 ug/ml 305 uM
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Dilute concentrated primer stock to 10 uM stock.
You have 305 uM stock you want 10 uM
stock. Divide what you want by what you
have 10 uM/305 uM 0.0328 3.28/100
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29 nmol/290 ul 0.1 nmol/ul 100 umol/l
100 uM
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25-nucleotide primer (50 GC 100
complementary to template) PCR contains 100 mM
NaCl What is melting temperature (Tm) of
duplex DNA formed between primer and template
DNA? Tm 16.6 log Na 0.41 ( GC) 81.5 -
500/bp Na molar salt concentration GC
percentage (whole number 50 50) bp
length of DNADNA hybrid in base pairs. Tm
16.6 log0.1 0.41 x 50 81.5 500/25
16.6 (-1) 20.5 81.5
20 65.5oC
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Test of PCR Reagents
bp
template added - no-template control
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Fermentas 100-bp Gene Ruler Plus DNA ladder
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