Dr. Duncan Clark
duncan at genesys.demon.co.uk
Thu Oct 2 05:54:34 EST 1997
In article <5jgt7m$orh at cardinal1.Stanford.EDU>, John Ladasky
<ladasky at leland.Stanford.EDU> writes
>I have found that two things have greatly increased my ability to
>do long PCR. First, use HPLC-purified primers.
Also use very high quality nucleotides. The presence of trace levels of
dUTP is enough to stop long PCR above say 10kb on lambda by inhibiting
the proof-reading component.
We find that Taq on its own will amplify 10kb of lambda, albeit poorly,
and that a 10kb PCR of lambda is equivalent to PCRing 2kb of human
genomic. Beoyond that you have to use mixes. Mg optimum for anything
longer is very narrow so use 0.1mM steps in the optimisation. We've
found that even unpurified primers will work up to 35kb so that may not
be that critical.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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