>"Re-transform isolated pure clones without a problem" Does this mean you can isolate both the new and parental plasmids from your mixes? If not, I suspect you have duplicate regions in a single plasmid. We have seen this numerous times with quick change. Not sure the mechanism, but the result is often as if the primers were ligated end to end.
On Wed, Apr 24, 2013 at 11:36 AM, Pow Joshi <[email protected]> wrote: > On 24 April 2013 12:55, Vladimir Gainullin <[email protected]> wrote: > > > 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? > > > > > > > The possibility of concatamers? I understand that they are possible is > various situations of cloning. > > Pow > > > > 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 > > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
