No ligation of PCR product in pGemT-Easy

Duncan Clark junk at hgmp.mrc.ac.uk
Wed Jul 30 06:30:32 EST 2003


Historians believe that in newspost 
<1059515678.3f26ed1e925e4 at webmail.nmsu.edu> on Tue, 29 Jul 2003, Dr. 
Hiranya S. Roychowdhury <hroychow at nmsu.edu> penned the following 
literary masterpiece:
>BTW, I am not sure that the vectors come phophorylated (as suggested by
>Duncan).

As far as I am aware all non-TOPO Invitrogen TA vectors are ready for 
use without having to add any additional terminal phosphate by kinasing 
with T4 PNK and ATP. They work for us with just ligating in PCR product 
straight from the PCR itself i.e. no clean up.

TOPO vectors should behave exactly the same.

If you kinase the PCR product or use primers synthesised with a 5' 
phosphate you stand the chance of generating multimeric inserts. Now I 
know the PCR product, in theory, has a single base overhang and 
shouldn't ligate to itself, but you are dependant upon having that 
overhang present in the first place, which is itself dependant to some 
extent (old Biotechniques reference) upon the primer sequence. Of course 
if it doesn't have an overhang and ligates to itself, it really 
shouldn't then ligate to the vector with an overhang and so on ........

Whatever, TA cloning works :-)

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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