another PCR question - (Mar/26/2006 )

Hi,
Is it necessary to heat the PCR reaction mixture containing DNA template, water, primers, Pfu buffer, dNTP mix at 95oC for 2 minutes and put on ice before adding Pfu polymease? What is the purpose for doing this?
Thanks.

I don't know if it should be done like this but I never do it. I just add all together, pt it in PCR machine and heat it at 92C for 2 mins as initial denaturation so that DNA strand seperates. Thats all I know the reason for running it at 92C for 2 mins, If anybody else has dencent answer, please post.

Hi
I did some pcr using hot-start but it did not effect the pcr much.But i did it such a way that after first denaturation 94 C for 3 minutes (or so) stop the thermal cycler and add the polymerase(or primer, template ).

Most enzymes are stable enough to have plenty of activity left after initial denaturation for all subsequent cycles.

If you decide to denature @ 95 for 2 minutes (or any other initial denaturation) without the enzyme in it, you ineed have to put your mixture on ice directly, otherwise your template may renature...

Pfu is very heat stable, and so you can denature at 96 or even 98 (at the initial denaturation step and during the cycling steps. You can do this for only a few seconds per cycle (say 15 or 20 seconds). Also unlike Taq (which has some) Pfu has virtually no activity at RT so in fact you kinda automatically do a hot start with Pfu polymerase. Then i am not sure about a hot start with things like Genomic DNA. The DNA is double stranded (if you did not denature it for any reason before the PCR) and so there is no where where the primers can bind (it is as it where self protected!) before your first denaturing step. Were you to start with a single stranded template using Taq i can see that a hot start could be benificial to reduce back ground.

try phusion polymerase, even less errors and (usually) much more product.

unfortunately at the moment I'm not able to buy anything new but thanks anyway