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

Dr Alexander J Annala ucklw08 at ucl.ac.uk
Sat Mar 30 02:48:31 EST 1996


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
  






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