Re: Plasmid mix in the clone - the mechanism?

[Vladimir Gainullin]

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?
>
> DK
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[Pow Joshi]

The possibility of concatamers? I understand that they are possible is
various situations of cloning.

Pow

[Dunowska, Magda]

Here is a link to a discussion on the topic that I found useful at the time we seemed to have a similar problem (mix of plasmids from one plasmid prep): http://bitesizebio.com/articles/plasmid-retention/

Magda

________________________________________
From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of Pow Joshi [pow....@gmail.com]
Sent: Thursday, April 25, 2013 7:36 AM
To: Vladimir Gainullin
Cc: met...@magpie.bio.indiana.edu; DK
Subject: Re: Plasmid mix in the clone - the mechanism?

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[Jonathan Rupp]

>"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.

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