PCR Subcloning with restriction sites

Tony Schountz tschount at mesastate.edu
Sun Mar 26 11:04:43 EST 2000


We are interested in subcloning a gene from pGEM-T into pET-23b. Our
strategy was to use PCR to amplify from the pGEM-T template, but with
modified primers that included BamHI on the 5' end of the forward primer
and XhoI on the 5' end of the reverse primer and clone again into
pGEM-T. This presumably would give us these restiction sites and allow
easy subcloning into the pET vector in-frame. However, after doing the
PCR and cloning back into pGEM-T Easy, we found that BamHI nor XhoI
would cut the plasmid, although the pGEM-T Easy EcoRI sites adjacent to
the cloning site digested fine. Our enzymes are good, so I'm not sure
what could account for these difficulties.

I'd appreciate any suggestions or work arounds to this problem.

Thanks,

Tony

--
Tony Schountz, Ph.D.
Department of Biological Sciences
Mesa State College
mailto:tschount at mesastate.edu






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