Lab 2: PCR Flashcards
PCR
Method to selectively amplify a particular DNA either from small quantities or from among a large population of DNA. Sensitive enough to amplify single molecules.
Goal of experiment
Amplify G0alpha cDNA from rate heart library.
PCR theory
Denature dsDNA at high temp (94C), anneal primers to template DNA at lower temp (55-65C), polymerization/elongation of new DNA strand using a thermostable polymerase recognizing primer template hybrid (Taq pol from Thermus aquaticus bacteria) synthesizes new DNA strand incorporating new dNTPs in 5-3 direction (72C).
What are some other polymerase a used and what is ideal for a polymerase?
Pfu, Vent, Deep vent, Pfusion). Best is to have high fidelity (proofreading= fewer errors) and high processivity (high number of nucleotides incorporated).
How much is the sequence amplified?
Long product grows linearly, # of copies = 2n. PCR target grows exponentially, # of copies= 2^(n-2)
Quantitative PCR
Basic PCR is not quantitative-exhaust the nucleotides. Real time PCR is quantitative. In real time PCR, the amplified gene is detected as it’s being made.
What are some considerations about primers?
Specificity- specific for the intended target sequence (avoid non specific hybridization): uniqueness, length, base comp, avoid self annealing.
Stability- form stable duplex with template under PCR conditions: annealing temp, matched PCR conditions for the primer pair.
What are some basic principles of primer design?
Primer length: usually 17-22 nts.
Base comp: G/C content should be around 50%, avoid runs of three or more identical nucleotides, 3’ G/C clamp.
Melting and annealing temps: matching primer melting temps (Tm), annealing temp should be 5 C lower than primer melting temp, preferably Tm of 55-65C.
Avoid secondary structures: hairpin loops and dimers.
Design primers from unique (not repetitive/ not palindromic) sequences.
Why do you want a 50% GC content?
GC has 3 hydrogen bonds between the strands rather than 2 with AT, so it’s stronger.
What are some important PCR parameters?
Primer design, annealing temp, DNA template quality, DNA POL, magnesium cofactors required for DNA pols, pH=7-8, fresh dNTPS and enough of them (they slowly hydrolyzed at -80C), extension time= 1 min per kb of product to be amped (72C), number of cycles: depends on the quality of template DNA and is usually 25-30.
What does buffer P1+ RNAse have?
Contains EDTA to chelate divalent cations to inhibit DNA by nucleases.
What does buffer P2 have?
NaOH and SDS which lyse bacteria.
What did buffer N3 do?
Neutralizes NaOH and high salt from sodium acetate helps precipitate out protein, chromosomal DNA, debris, and SDS. Plasmid remains
What did buffer PE have?
(70% EtOH)
What did buffer EB do?
Eluted DNA from column. Had tris-Cl
