selfmade TA cloning vectors
Mr. G. Morley
gmorley at hgmp.mrc.ac.uk
Tue Sep 15 06:51:07 EST 1998
Re: Re: self-made TAvectors
15 Sep 1998 01:58:47 -0700
BIOSCI International Newsgroups for Molecular Biology
---- 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.
I remember making these things ages ago...can t recall the entire protocol
but you certainly don't CIP the vector..after blunt end ligation you have
to incubate the cut vector in TTP with an enzyme to put T's on the end of
it....after this the vector should not re-ligate to an y great extent..
not very helpfull I know but I did this a Looooooonnng time ago..
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