rolf at lx.student.wau.nl
Thu Mar 27 20:32:34 EST 1997
Hi Monica Ruiz-Noriega,
Are the SmaI/ApaI sites at the complete end of the 2.1k fragment? Most RE's need
some additional bases or need much more time. Moreover, ApaI and SmaI are not
really nice RE's, they require lower temperatures (30C) and give problems with
some methylations (although I doubt that's the problem ;). If you suspect the
RE's not to work on the PCR fragment you can always try to clone the fragment
in 'your favorite bluntend/TA-overhang vector', and cut the fragment out with
the two enzymes. At least you'd be sure the 'cutting' worked.
> 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.
> Monica Ruiz-Noriega
> Susan Henry's Lab
> mur6+ at andrew.cmu.edu
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