Long PCR

Colin Rasmussen colin at nospam.fungus.usask.ca
Fri Jul 31 12:19:10 EST 1998

I need some feedback from anyone with experience in doing PCR of large
fragments (4kb).  We've just made a mini-library in pGEM using S. pombe
genomic DNA in order to clone the 5' end of a gene which we have a
complete cDNA for.  The sequence is not yet in the database so I can't get
it that way.  Anyways, we've cloned HindIII digested genomic DNA in pGEM
and we've made a midi-prep of DNA following transformation into E. coli. 
Using primers for a smaller fragment derived from the cDNA sequence (900
bp) we can show the presence of the fragment in the mini-library.  What
we'd like to do is to PCR from the FWD or REV primers of pGEM to a site at
the 5' end of the ORF.  The distance is about 4 kb, and I was wondering
what kinds of parameters we should set-up for the PCR.

An email reply would be greatly appreciated.  Thanks.

Colin Rasmussen
University of Saskatchewan

colin at fungus.usask.ca

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