PCR cloning

Monica Ruiz-Noriega mr6h+ at andrew.cmu.edu
Thu Mar 27 17:16:45 EST 1997


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 



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