In article <xjia-231096145648 at summers.mmg.uci.edu>, xjia at uci.edu (X Jia) wrote:
> can anyone give me some advises for my PCR on a piece of DNA that contains
> about 70% G/C? I have a lot of troubles to make the PCR working.
> thank you very much
> X. Jia,
>Xjia at uci.edu
Yes, maybe, just maybe I can be a little help.
High GC means that you will have a harder time separating the two strands
during the melting step at 94 degrees. Typical denaturation conditions
are 95 for 30 sec, or 97 for 15 seconds. Yet, for G+C rich templates
higher temperatures may be appropriate. If you do not completely melt
your DNA, it will snap back and lower your DNA yields. Be sure that you
don't overcook your taq though....the half-life of over 2 hours at 90
degrees drop dramatically when you get closer to 97. Good luck,
Barry at sccs.swarthmore.edu
PS: let me know if this helps at all. or if you have any questions.