Help!!!! how to clone a PCR fragment?

Mad Dan Eccles rpgrant at molbiol.ox.ac.uk
Wed Aug 9 05:01:38 EST 1995


In article <Pine.AUX.3.91.950803180003.17417A-100000 at mail.med.cornell.edu>, hmmoss at MAIL.MED.CORNELL.EDU (Heidi Moss) writes:
> I have a cloning project that involves inserting a PCR fragment with one 
> cohesive (Bam HI) and one blunt end into pBS.  I can foresee two problems 
> with this, namely: 1) the presence of the nontemplate specific A residue 
> that is added at the end of PCR fragments, which gives me a non-blunt 
> end, and 2) the absence of the 5' phosphate of the primer on the "blunt" 
> end.  I know the theory about what should be done to eliminate these 
> problems, but what I need is someone with a little experience doing 
> this.  I'll start with the nontemplated A issue: I've heard that 
> incubation with DNA pol I will "polish" the end by the activity of the 

My advice would be to use Pfu polymerase which does not leave an extra 
residue.

Secondly, if you don't phosphatase your vector, you won't need to kinase the 
insert.  If both vector cuts (BamH1, and eg SmaI for blunt) are efficient, you 
should not get high background.

Richard



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