PCR Flashcards
What happens removing MG2+ ions from mixture?
Increase Stringency
Too few — no product
Too many — increased mispriming
What happens with increased temperature?
Increased Stringency
Heat keeps primer and target separated; only hybridize with high compatibility
What happens with increased probe length?
Increased stringency
Less likely random sequence matching will occur
What happens with increased probe complexity?
Increased stringency
GC vs AT affect hybridization conditions
PCR conditions should be optimized around primer complexity
What happens with lower salt concentrations?
Increased stringency
In general, lower salt favors longer products; high salt favors shorter products
What happens with less time?
Increased stringency
Less time for unwanted binding
What happens with the addition of formamide?
Lowers stringency
Denatures to remove secondary structure of DNA, more readily available to primers
What happens with the addition of BSA?
Lowers stringency
Stabilize enzymes and bind inhibitors
What happens with increasing probe concentration?
Lowers stringency
More probe will lessen hybridization times
What are PCR inhibitors?
Hemoglobin
Heparin
Xylene
Phenol
Cyanol
Bromophenal blue
What are preferred blood tubes for PCR?
Lavender — EDTA
Yellow — ACD
Special blood tubes for DNA isolation?
Plasma preparation tube — PPT
Separates lymphocytes/granulocytes
What is the equation for DNA concentration calculations?
A=Ebc
A — absorbency
E — molar absorptivity
b — path length
c — concentration
Tm
4(G+C) + 2(A+T)
