Sequencing from E.coli.

[Zophonias O. Jonsson]

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.

Try it if you dare :)

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________