"LONG" RT/PCR

[Elizabeth Estabrook]

Dear Shahram and Steve

In response to Long PCR and particularly Shahram's reply:

: For denaturing such a large fragment you need at least a minute of

I am not sure about the need for at least a minute for large fragments 
and my only experience in this is that I recently attended a seminar 
given by a PE person regarding long PCR which he defined as greater than 
5 kb. They of course have a kit for long PCR in which they optimize for a 
fragments larger than 10 kb using lamdba cloned inserts and human genomic 
DNA. I have no affiliation with PE of course and I am not trying to get 
you to buy the kit.

One of the points that he stressed in the seminar was a short 
denaturation time: 5-10 sec at 95 C. Shorter times of denaturation always 
seemed to work the best supposedly because of the problem of potential 
heat damage to the DNA (depurination?). But one could also say that this 
time must be dependent on your machine and tubes.

In my own PCR work (keep in mind however that my largest PCR product I 
make routinely is only 2.5 kb) I have shortened my denaturation time down 
to 30 sec and comparision to my old denaturation time (1 min) showed the 
shortened time to be better (ie more product).

But I am curious what other people think of as an appropriate 
denaturation time?

Elizabeth
emestabrook at ucdavis.edu