Culture of E.Coli (SURE) / Large Plasmids / Foreign Sequences

Sultan Kalifa rufer at novsrv1.pio1.uni-heidelberg.de
Tue Apr 2 11:19:55 EST 1996


In article <4jq6b7$hfd at news1.h1.usa.pipeline.com>,2 Apr 1996 03:19:03 
GMT,mkoch at usa.pipeline.co wildly stated/shouted/said...
>
>On Mar 30, 1996 07:48:31 in article <Culture of E.Coli (SURE) / Large
>Plasmids / Foreign Sequences>, 'ucklw08 at ucl.ac.uk (Dr Alexander J Annala)'
>wrote: 
> 
> 
>>Sambrook/Frisch/Maniatis Molecular Cloning page 1.21 suggests that 
>>where plasmids grow poorly because of their large size or because of 
>>foreign sequences they carry it may be worthwhile to consider some 
>>alternative ways to grow and treat a bacterial culture. 
>>I have a BlueScript SK- derived plasmid containing a 12 KB mammalian 
>>intron which yields very small amounts of miniprep DNA (from 2 ml TB 
>>overnight cultures 50 ug/ml ampicillan).  The culture also appears  
>>to be unstable in the sense that nothing grows in a new culture tube  
>>(2 ml TB with 50 ug/ml ampicillan) innoculated with a sample drawn 
>>from the previous culture.  It would seem the cells no longer harbor 
>>ampicillan resistance. 
>> 
>>What alternative ways to grow and treat the bacterial culture should 
>>be considered in the instant situation? 
>> 
>>Thanks, AJ Annala 

may i humbly suggest our publication in biotechniques entitled:
"improved expression of toxic proteins in e.coli" ?
the suggested "plating" method may very well solve your problems, 
as it concerns itself with extremely instable plasmids.

biotechniques, vol. 19, number 2, 1995, Suter-Crazzolara and Unsicker


























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