Re-Transformation of BAC-plasmid-Help needed

Martin Messerle Messerle at lmb.uni-muenchen.de
Thu Sep 19 12:43:32 EST 1996


Dear Netters,

I got a very nice BAC-plasmid of about 230 kb. For 
mutagenesis I have to retransform the BAC into a
different E.coli strain. The electroporation 
efficiency is very poor. Furthermore, up to now I
always ended up with much smaller plasmids after 
retransformation, even when I retransformed the
plasmid into the original E.coli strain DH10B. I 
isolated the BAC plasmid by alkaline lysis followed
by phenol/cloroform extraction and EtOH 
precipitation. I tried to handle the DNA as gentle 
as
possible (cut tips etc.) and expected a high 
percentage of supercoiled DNA.

Any helpful suggestions?
Thanks a lot.

Martin Messerle
Max v. Pettenkofer Institute for Virology am 
Genzentrum
Muenchen, Germany
EMail: Messerle at lmb.uni-muenchen.de




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