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In article <[Only registered users see links. ].net> , malihe keramati <[Only registered users see links. ]> wrote:
In about 10 years, I had only one case like this. The solution was:
extend at 65C instead of 72C. The gene in question had stretches
of ridiculously high AT (over 90%). None of the conventional additives
worked but changing extension temp did. Try it, you don't have a lot to
In general, I wholeheartedly agree with the recommendation of
PfuFusion. In terms of fidelity and processiveness, it has so far
no equal (that I know of).
To Peter Ellis:
First to come to mind:
1. You can use it for efficient blunt end cloning without extra steps.
2. You can save money by not sequencing insane number of clones
if your insert is ~ 5 kbp. In fact, if you can take P=0.05 (not that I'd
recommend it), you can just skip sequencing altogether.
|pcr , pfu|