Long Range PCR Question

Dr. Duncan Clark duncan at genesys.demon.co.uk
Thu Apr 18 03:41:21 EST 1996


In article <4l2gjm$c6f at mserv1.dl.ac.uk>, KONSTANTIN.KANYUKA at bbsrc.ac.uk
writes
>Hi Bionetters,
>
>
>Do you know of any good and reliable commercial PCR Kits
>allowed to aplify fragment ~20 Kb from genomic DNA?
>I would also appreciate any suggestions, tips and tricks.

One tip is to be careful of your choice of dNTP's. We have found that
for up to 15kb any dNTP's work. Above that we had real problems just
amplifying 20kb from lambda and it was due to dNTP source. I could show
that one very well known suppliers dTTP was killing the PCR completely.
The consensus from a few sources is that the proofreading enzymes in the
mixes are very sensitive to dUTP contamination in dNTP's. I don't know
if dUTP was the offending contaminant. The offending nucleotide looked
OK on FPLC but the proofreading enzymes are sensitive to ppm
contaminants so it is unlikely to show up on FPLC. 

Perkin Elmer's dNTP's worked as did Boehringer's.

Duncan  


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