Sequencing from E.coli.

Zophonias O. Jonsson zjons at
Thu Nov 16 02:59:37 EST 1995

In article <48dro1$5jv at>,
salger at (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 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

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