PCR cloning

Yvon CAVALOC cavaloc at sun-recomgen.univ-rennes1.fr
Fri Mar 28 07:02:01 EST 1997


Monica Ruiz-Noriega wrote:
> 
> Recently I've been trying to clone a PCR product but my attempts haven't
> been succeful. Do I need to keep anything in mind when doing so?.
> What I did: PCR out a 2.1 kb fragment with SmaI and Apa I sites at the
> ends. Phenol-chlorophorm, ethanol precipitate my fragment and cut wiht
> the enzymes mentioned above. Then I just tried to ligate this product to
> a linearized plasmid cut with the same enzymes. Before trying the
> ligation reactions I checked both DNAs by electrophoresis and looked
> fine (not degraded and in the case of the plasmid, looks cut)
> I cannot get any E. coli transformants. It is the first time that I
> subclone PCR products and I do not know if I need to do anything
> different. The ligation reactions that I am using have always worked
> before but they're not working now.
> Any suggestions and comments will be highly appreciated.
> Thanks
> Monica Ruiz-Noriega
> Susan Henry's Lab
> mur6+ at andrew.cmu.edu

I also had some troubles to cut PCR product close to the end although I
left 4 nucleotides after the restriction site. An easy way to check
whether the digestion is the reason of your problems is to ligate your
digested fragment on himself: If both extremities are well cut, you will
see a smear above 4-6 kb.

Yvon



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