Patrick F.H. Lai the Graduate Student
pfhlai at LOOKSMART.COM
Tue Sep 15 03:58:47 EST 1998
---- Begin Original Message ----
Markus Schneemann <schneema at cmgm.Stanford.EDU> wrote:
> Hi all:
> I'm about to generate my own TA-vector for PCR product cloning.
> I'll digest pXcmkn12 with XcmI. But then I do not know whether I have to
> dephosporylate in order to prevent religation.
> Has anybody of you done that before ?
> Did you use Boeringer's shrimp alkaline phosphatase (easier to deactivate
> later) or any ordinary CIP ?
Since your PCR product won't be phosphorylated (unless you phosphorylate
your primers first), you shouldn't CIP the vector... not to mention of
course, the T-Tailed vector should not religate because the overhanging
nucleotides won't anneal to each other anyway.
---- End Original Message ----
How about CIPping the vector and phosphorylate the PCR product with
PNK ? Would the extra work improve the cloning efficiency ?
Is it worth it ?
Just a thought...
or keep looking.
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