PCR Subcloning with restriction sites

John Thompson 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.

John Thompson
Merck Research Labs

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