Post-ligation restriction

Klaus Salger salger at zi.biologie.uni-muenchen.de
Fri Jun 28 13:29:31 EST 1996


Jean-Paul CADORET, Gie-ra Montpellier PDG-DRV-RA-DRIM, +33-6714-4707
wrote:
> 
> Hi netters,
> 
> Could somebody give me an explaination to this:
> 
> I blunt ligated a 1300 bp PCR insert using PCR script from stratagene.
> Everything went OK with white colonies and so on.
> After a midi prep of 4 colonies and restriction analysis, several restriction
> sites are definitively reluctant to cutting. I checked the enzyme temperature
> as well as buffers. It seems that this appears more on one side of the MCS.
> Is the vector corrupted?
> by my ligations?
> Had somebody that broblem once ?
> 
> Thanks
> JP

What enzyme did you use to blunt-cut your vector?
Especially SmaI (or a contaminating nuclease?) is said to nibble back
the ends of the DNA.
Anyway, if you want to check for exonuclease digest of your cut vector
you could just religate the vector (without insert) and check for the
presence or absence of the RE sites with some clones.
If some sites near the cutting site are missing you might try to do the
digest with less enzyme for a shorter time, eventually at a lower
temperature (with cleaner DNA?).

Cheers
  Klaus

-- 
Klaus Salger                phone : +49 (0)89 5902 -502
Zoologisches Institut       FAX   :                -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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Germany

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