PCR cloning

Sai siyer at bmg.bhs.uab.edu
Thu Mar 27 19:17:41 EST 1997


In article <EnCj7Bu00iWl09Q3w0 at andrew.cmu.edu>, Monica Ruiz-Noriega
<mr6h+ at andrew.cmu.edu> 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

if you used taq polymerase, then it is most likely that your pcr product
has 3' dATP overhangs.  taq will often do this and cannot "undo" it cuz it
lacks its 3'-5' proofreading exonuclease activity.  now since you digested
it with the above mentioned enzymes, that is most probably not your
problem.  if you took this to the next trouble shooting level, then the
question would be if you incorporated the extra random bases that's needed
for the above mentoined restriction enzymes in your pcr product.  there
are some enzymes that need at least 5 or 6 extra bases preceding the site
of cleavage.  Nco1 is one enzyme that behaves this way.  these extra bases
can be anything, but they must be there.  so you should probably check the
stats on your enzymes and see if they need extra bases.  if they do, then
re-design pcr primers with the extra bases , re-PCR and try it again. 
that should hopefully do the trick.  good luck...

-- 
Sai Iyer (sai at uab.edu)
Pre-doctoral fellow i.e lowly graduate student (Dr.Gerald Hart's lab)
Dept of Biochemistry and Molecular Genetics
University of Alabama at Birmingham...



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