PCRing a large gene

Bernard Chong Ev Khoo bcek1 at mbuk.bio.cam.ac.uk
Mon Feb 14 05:14:07 EST 1994


Dear all,

OK, I know that this is probably an FAQ, but the relevant articles have just
been cancelled at my site, so,

I'm going to try to PCR up a 3.1kb gene from Saccharomyces cerevisiae genomic
DNA. Now, since it's so long, my concerns are:
(1) whether this will be efficient
(2) accuracy -- the clone is for expression

I have some Taq polymerase and some VentR as well. Reading through the tech
specs on VentR, it advises careful optimization of Mg, primer levels, dNTPs
and enzyme levels. Could anybody who's ever used VentR advise me as to the
optimal levels of these parameters? Or will Taq be good enough if I decrease
Mg to 1-1.5mM and keep the dNTPs at a fairly low level (200-250uM)? Not
forgetting to keep the number of cycles low, of course...

Otherwise, it's back to designing oligos to PCR up bits of the gene...

Bernard Khoo
Wellcome/CRC Institute
University of Cambridge, UK.

bcek1 at cus.cam.ac.uk
     @mbuk.bio.cam.ac.uk

Disclaimer: My opinions only, not those of my employer or of my school.



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