Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Thu Oct 5 09:08:17 EST 2000
In article <8rhe4v$qp0$1 at news.net.uni-c.dk>, Ali El-Salanti
<isl40117 at worldonline.dk> writes
>But I can not Pfu amplify the large sequnces.
>Maybe it should be noted that the template i 80 % AT.
I think this may be a cause of your problems.
It may well be that your extension temp of 70-72C is actually too high
such that your product is melting. (Reported in an NAR many many moons
ago and this was their solution)
Try an extension at 60C and make sure you have a low enough annealing
temp if your primers are also AT.
If that still doesn't work then you may have to try a Taq:Pfu mix.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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