PCR problem: pfu working selectively?

L Celeste via methods%40net.bio.net (by celesteel from gmail.com)
Tue May 26 16:15:35 EST 2009


Hi everyone,

I have been trying to amplify a 9 kb pBAC fragment with Phusion Flash Master
Mix. Although we didn't get the right bands, but we got bands (one at 7 kb)
at annealing temperature of up to 60oC and extension temperature of 72oC.
But then when we switched to Pfu with annealing temperature of 60 oC and 61
oC, and extension temperature of 68 oC we were not able to produce any bands
at all. Thinking that our Pfu might not be working at all, I repeated this
experiment along with another experiment (target fragement 11 kb) at 55 oC.
We got bands for the experiment with target fragment of 11 kb, but again no
band for the orginal pBAC experiment.

Any advice would be appreciated.

Thanks
Celeste


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