PCR Subcloning with restriction sites
jrt at home.com
Sat Apr 1 21:57:19 EST 2000
Tony Schountz <tschount at mesastate.edu> wrote:
>If I designed my primers with the first 6 nucleotides for restriction sites, then 20
>nucleotides that are gene specific, then the result after PCR (Taq) should have
>amplicons with the restriction sites at the very end with an A overhang on the 3'
>ends. When this was cloned into pGEM-T, I expected that the restriction sites would
>have been preserved.
If the PCR goes cleanly and efficiently all the way to the end.
Add the 6 extra bases on the end that people are suggesting would give
you a little buffer zone and you could also save a step by cutting the
PCR product and cloning directly into the pET vector.
>Perhaps I should have the plasmid sequenced to see what
>happened to the restriction site?
That would indeed nail it. I'll bet the sites aren't there.
Merck Research Labs
More information about the Methods