Choosing PCR conditions

Question

How does one choose PCR conditions? Does it depend on the taxon, on the DNA concentration, on the primers or anything else?

Answer 1

You need to first build an initial protocol and then optimize it.

The most important to determine the conditions is the polymerase used. Some polymerases work at higher or lower temperatures and will work faster or slower. Find the default protocol from the polymerase seller and this will give you a good idea where to start.

The second most important is the primers used. Depending on the GC % in the primers and their length, the times or temperature of each step will change, or if you prefer will need to be Optimized. The length of the DNA fragments will also determine the length of the cycles. Most polymerase sellers will includes steps to optimize the reaction depending on the quantity of DNA used, primer length and other parameters.

For more information:

Designing primers:

Taq DNA polymerase protocol example:

Optimizing PCR

• PCR thermal profile, Deoxynucleoside Triphosphates and Primers are interesting paragraphs

Answer 2

Use primer3 to design your primers, then just use whatever protocol your lab usually used for PCR.

A high annealing temp solves many problems, and a good enough taq won't mind.

I successfully sanger sequenced thousands and thousands of mammalian exons, with primer3 designed primers, a HotStar Taq, and a 60 degree annealing temp. Worked almost every time.

Obviously if you are doing long PCR, or working with a very high GC or low GC species, that might requires more optimizing.

Answer 3

According to my knowledge the main thing in annealing temperature in PCR Primers annealing temperature should be selected by keeping in 5° less than the lower melting temperature of primers Secondly MgCl2 working Concentration also plays a major role in PCR optimization