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
Wow. That one always generates a slew of responses. In my hands glycerol
has not been
much help. Don't know why.
Here's what I've done with 70% GC templates: use 7.5-10% DMSO. Use a "Hot
Start"--adding the enzyme after cooking your template @99 degrees for a
few minutes. Then lower the temp. to 90° and add the enz. Use a high temp
denaturation and don't anneal the primers too long. Begin cycling with the
95° denature step.
Recently, I had some template that still wouldn't respond until I did all
of the above with a cocktail that was 50mM TMAC (tetramethylammonium
chloride). See Chevet, et.al., 1995, NAR 23. #6, pp.3343-3344. Worked so
well I was able to skip the hot start. Those guys saved my bacon!
Let me know how you do-and welcome to the "Nobody Can Amplify This Crap" club!
Sorry if this is posted twice. I forgot to sign the first post and may not
have aborted it in time.