steve.roberds at ibm.net
Fri Jul 31 23:07:11 EST 1998
I've used Boehringher Mannheim's Expand kit (a mix of Taq and Pwo
polymerases). Since your library is circular, use their instructions for
cDNA (with buffer 3). We've used it up to 11 kb, and it should go much
Because your vector-specific primer will bind to everything in the library,
you may need to adjust the primer concentration, and maybe the annealing
temperature to optimize specificity. But I'd try the kit guidelines first
and see what you get. Good luck.
Colin Rasmussen wrote in message ...
>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.
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