DpnI digest?
On Wed, Apr 24, 2013 at 12:39 AM, DK <[email protected]> wrote: > Wonder if anyone has good explanation as to how this is occuring: > > Once in a while, looking at sequences of mutant clones obtained by > "QuikChange", we'd see a simulateneous presence of both parent > and mutated genotype. > > I always discounted it on "cryptic double clone" or simple screw ups. > The frequency was never something to really pay attention to. > > Until this last time. I've screened clones by colony PCR, picked > positives, sequenced two clones. In BOTH clones, it is definitely > a mix. A simple mix up can be confidently excluded in this case > (re-tested, double checked going back to the original colony PCR > templates, etc, etc). > > Re-transform isolated pure clones without a problem but it's got me > thinking about the mechanism. Why would two different plasmids > end up in the same cell and persist together all the way through > a large clone and an overnight culture grown from it? And, how > to control/limit this occurence? With two clones having the same > wierd problem, this looks like something systematic that's worth > understanding in order to avoid... > > Technical details in case they matter: the mutagenesis was done > for two sites simulatenously - one was just a point mutation and > another was 60 bp insertion over deletion of the 66 bp. Both were > done with primer against the single strand, a la "QuikChange Multi". > It's only the long mutation that's a mixture - point mutation is > homogeneous. The % of double mutants by cPCR was low, 3/18. > This was the first time I used chemical cells for QuickChange, > having done exclusively electroporation before - wonder if that > can make a difference too. > > Any opinions? > > DK > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
