help: c oning PFU pcr fragment

Patrick F.H. Lai the Graduate Student pfhlai at LOOKSMART.COM
Tue Sep 29 14:00:28 EST 1998


---- Begin Original Message ----
Mr. G. Morley wrote:
> Hi all,
>                 I am having trouble cloning a some PFU pcr generated
> products. This enzyme should produce a blunt ended fragment which
> I have kinased. However it repeatedly fails to clone into a blunt
> ended vector that has been CIP treated. Any suggestions? the fragments
> are purified after kinase treatment by removal from LMP gel. Do the
> fragments require phenoling?
> Any suggestions would be appreciated as it is driving me nuts.
> 
> Thanks in advance.
> 
> Gary morley
> g.morley at ucl.ac.uk


---- End Original Message ----

Hi Gary and Netters,

How about T-tailing the blunted vector and A-tailing the Pfu-generated insert with Taq Polymerase in 72 deg C for 30 min ?
They should ligate okay without all the troubles with CIP and PNK, I think.  Right ???




Hope this is useful info.   :-)


Patrick F.H. Lai  < PFHLai at looksmart.com >
Graduate Student
University of Toronto
Toronto, Ontario, Canada

P.S.  I am merely a GradStudent.
One should take a HUGE grain of salt with whatever I have typed above.
Good Luck.   :-)



 


 
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