Cloning long PCR roducts

Bresagen bresagen3 at adelaide.on.net
Wed Jun 18 10:15:00 EST 1997


I have tried any number of techniques to clone long PCR products over
the last year, and had very limited success.
I've tried pCR-Script, pGEM-T, and even cutting at internal sites (ie.
at sites that are 200-300bp in from the primers) doesn't seem to help.
Probably the most successful approach has been with pGEM-T, but it's
still hard work to get a clone. These products just seem to be
incredibly hard to clone compared to normal PCR.
Has anyone with significant long-PCR experience got any comments or
advice? 
The PCR reactions themselves work well. I routinely amplify 6-10kb from
mammalian genomic DNA with a Taq:Pfu (80:1) mix.



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