Antibodies from gel slices AND pGEM-T mutations

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Apr 22 08:30:55 EST 1998


In article <353D076E.6477 at chugaibio.com>, Dom Spinella
<dspinella at chugaibio.com> writes
> For what its worth, in our hands the
>best fidelity is achieved with PE's "Ultma" polymerase (although at some
>expense in product yield and PCR "robustness").


That's quite surprising as all published mutation analysis tests on this
enzyme report that it is worse than Taq! (NAR a couple of years or so
ago).

With regards the orginal problem from Reading. Use the minimum amount of
cycles possible with the maximum template. The more cycles you do the
more errors you will see. Maybe you are just unlucky and your template
really is error prone. As you are using pGEMT and presumably with TA
cloning, are all your clones in one direction. If your wt gene really is
semi-lethal, one would expect the gene to be cloned preferentially,
orientated against the lac promoter. If you don't see this then I would
suggest that is not the case. We find that anything that causes a slight
problem in E.coli always goes in opposite to lac in TA cloning.

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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