Reason for odd PCR conditions - (Dec/29/2010 )
Hello, I am running a PCR with the following conditions: 94C for 1 min, after initial denaturation, 94C for 15 sec, 55C for 30 sec, 72C for 30 sec, repeat 15x then 94C for 15 sec, 55C for 30 sec, 72C for 45 sec, repeat 25x.
I am amplifying off of plasmid DNA a 600 bp product. I am curious as to why I have one set of steps (repeated 15x) with an elongation of 30 sec and then another set of steps (repeated 25x) with an elongation of 45 sec. I am using Taq polymerase. The PCR works great, IO just don't understand the need for 2 sets of steps.
actually, i think 1 step is ok~ i also dont see there is any reason for separate it into 2 steps
longer elongation time just for synthesize longer DNA fragment~
May be this is a special technique (although i dont think so). Let see how other ppl answer
I've never seen this either. Where did you get this protocol?
I saw this type of PCR in a kind of mixed touchdown and gradient protocol. But then in the first part the annealing temperature is decreased with every repeat. The second part is then the actual amplification of the fragments that could be synthesised in the first part.
This is e.g. used in cross-species amplifications (primers of different species are tried out for a new one).
Anyway perhaps you overlooked the temperature decrease?