Sequencing from E.coli.

Rafael Maldonado rafael at howard.genetics.utah.edu
Fri Nov 17 19:25:06 EST 1995


On 16 Nov 1995, Zophonias O. Jonsson wrote:

> In article <48dro1$5jv at sparcserver.lrz-muenchen.de>,
> salger at wap1.zi.biologie.uni-muenchen.de (Klaus Salger) wrote:
> 
> > 
> > MC1061 doesn't carry the endA mutation. It seems to be difficult to get rid
> > of this endonuclease which makes sequencing very difficult. Switching to
> > an endA- strain (DH5a, XL1,...) should fix this problem.
> > 
>   If you isolate plasmids by alkaline lysis, heating for 10 min at 70*C in
> the NaOH step seems to do the job.  As a bonus most of the RNA is
> degraded.  The DNA will of course be severely denaturated but it sequences
> very well.  I manage to get decent sequences from plasmids treated this
> way even when I leave out the phenol/chloroform steps and precipitate
> directly with IsoprOH.

On the other hand, we sequence every day plasmids from a endA+ strain. 
Alkaline lysis, plus 30 min incubation at 37 with RNAse, and phenol 
extraction seem enough to overcome the endonuclease.

However, I have heard that other plasmid extraction protocols (as boiling 
method) are not suitable in endA+ strains.

Conclusion: use an endA- strain if it is possible. If it is not, use a 
phenol step.

	
	Rafa

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