On 24 April 2013 12:55, Vladimir Gainullin <gainul...@gmail.com> wrote:
> DpnI digest? > > > On Wed, Apr 24, 2013 at 12:39 AM, DK <d...@no.email.thankstospam.net> 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 > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
