PCR product cloning problems

Emir Khatipov khatipovNO at NOuchicago.edu
Thu Aug 15 15:43:47 EST 2002


I hope the problem is not that your primers are designed to exactly flank
the cloned fragments from both ends. In such a case the sequence file you
get from your sequencing facility will be missing a few bps from the ends,
simply because the facility manager would cut off bad portions of
chromatograms before converting them to the text sequence. Otherwise, either
your solutions (or solutions used at the sequencing facility) are
contaminated with exonucleases.
Emir

"Bill Rogers" <brogers at noguchi.mimcom.net> wrote in message
news:8984713a.0208140655.7e3f3ba5 at posting.google.com...
> I recently cloned and sequenced a batch of PCR products and found that
> many of the products were missing 1-10 bases from the ends. To me this
> could be either
>
> 1. lousy primers (and I'm checking that now)
> 2. exonuclease activity during the PCR reaction (I used the
> Boehringer-Mannheim core PCR kit, Taq polymerase)
> 3. exonuclease activity during cloning (using the PCR Script kit)
>
> Has anyone here had similar problems or have any suggestions to avoid
> this?
>
> Thanks for any help you can offer.
>
> Bill





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