Plasmid mutation in e. coli?

Anders Gorm Pedersen gorm at cbs.dtu.dk
Tue Aug 29 03:19:27 EST 2000


"Nicolas von Ahsen" wrote:
> >Can you sequence the PCR product directly? Are the errors still in that
> >rather than the individual PCR molecule you cloned and subsequently
> >sequenced.
> I'm not much used to that. We usually sequence plasmid DNA, I tried
> PCR products once and that wasn't fine.

On the other hand this is the only way you can really find out if the
problem is PCR or plasmid maintenance. In my experience sequencing of
PCR-products isn't so bad, just make sure to clean the PCR product prior
to sequencing, using one of the several spin-columns that exist (say,
Qiagen "Qiaquick").

> >This is the next problem. If you are cloning this cDNA downstream of a
> >strong promoter i.e. lac, maybe in reading frame such that you generate
> >some expression and your gene product is giving E.coli a problem then
> >the plasmid will re-arrange, delete or the gene will be mutated (not
> >sure of the mechanism but it happens!) until it can be maintained.

That mechanism could be selection for a few rearranged plasmids I guess?

> Interesting observation, but I'm using a mamalian expression vector
> and don't expect much to happen in e.coli. Also, the protein shouldn't
> be toxic.

I vaguely recall that repeats (direct or inverted) can cause lower
plasmid stability in coli. Could this be a problem?

> So the best way to go is probably more template, less cycles and less
> time in the e.coli.

If all else fails you might consider constructing the gene from scratch
using oligonucleotides?

Good luck!

Anders 
 
---------------------------------------------------------------
 Anders Gorm Pedersen, cand.scient., Ph.D.  (gorm at cbs.dtu.dk)

 Center for Biological Sequence Analysis
 Technical University of Denmark
 Bldg 208, DK-2800 Lyngby, Denmark

 phone: (+45) 45 25 24 84
 fax:   (+45) 45 93 15 85

 Web: http://www.cbs.dtu.dk/gorm/
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