fzestabr at bullwinkle.ucdavis.edu
Wed Nov 16 14:52:25 EST 1994
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
emestabrook at ucdavis.edu
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