shake after electroporation?
rafael at corona.med.utah.edu
Mon Mar 20 14:41:03 EST 1995
On Mon, 20 Mar 1995, kevin c. graham wrote:
> In article <3kkbcl$pd at mark.ucdavis.edu>, szcooley at chip.ucdavis.edu
> (Michael Cooley) wrote:
> > Qunfeng Dong (qfdong at iastate.edu) wrote:
> > : Hi,there:
> > : I used to shake cells after electroporation at 37C for 1 hrs, somebody
> > : told me it'd better not shake,just plate cells! the reason is that if cells
> > : grow quickly, the plasmid DNA could be kicked out of cells. I never heard
> > : this before, is it right?
> I would suggest that the 37 C recovery is helpful - its the Shaking part
> that may be adverse to the proceedure as you are trying to gain entry of
> you Dna into the cells - but if you treat them too harshly (shaking?)
> that this may not be of assistance. I've plated immediately and after half
> hour stationary incub. the latter certainly gives you better results but
> the former works when you're in a hurry!
The shaking always increase the recovery of the cells, and hence, the
number of transformants. But if your plasmid encode some toxic protein,
or it's unstable for some reason, you can choose not making the
incubation at all, and plate directly. Or if you only want one colony and
don't mind about high eficiencies!
You'll get less number of colonies, but it may work, specially if your
antibiotic used for the selection is bacteriostatic one (as tet) and
not bacteriocidal (amp, and the strongest, kan).
The shaking from a normal incubator won't be bad for your cells, IMO.
Rafael Maldonado "You gave to a boy the job
Department of Human Genetics of a man. He's dead."
University of Utah
Salt Lake City, Utah 84112. USA. John Wayne
Rafael at genetics.med.utah.edu
Rafael at corona.med.utah.edu "No por mucho madrugar, amanece
Tlf: 801-581-4429 mas temprano"
More information about the Methods