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 O. Jonsson
Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75
Universitat Zurich-Irchel Fax: (41-1)-362-05-01