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

Michael Koch mkoch at usa.pipeline.com
Mon Apr 1 22:19:03 EST 1996


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.  Presumably 
>this means alternative ways not included in the discussion which is 
>immediately above on the same page about chloramphenicol treatment. 
> 
>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. 
> 
>One naive approach might be to grow a somewhat larger culture (maybe 
>30 ml) in a flask to about 0.4 OD, innoculate with chloramphenicol 
>170 ug/ml (to inhibit protein synthesis - thus inhibiting chromosome  
>replication), and continue incubation for several hours to amplify  
>the plasmid containing the intron.  However, Maniatis et al suggest 
>this may not be the best approach for this situation. 
> 
>What alternative ways to grow and treat the bacterial culture should 
>be considered in the instant situation? 
> 
>Thanks, AJ Annala 
 
Try 400ug/ml Amp. 50ug/ml is prob. not enough, most coli strains 
are resistant to ~30-50 ug/ml. What you see is that you amplify a lot 
of cells that have NO PLASMID (ever see satelite colonies on Amp 
plates?), hence when subcultured after an extended period of growth 
nothing will grow in FRESH Amp media. MIKE...



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