Culture of E.Coli (SURE) / Large Plasmids / Foreign Sequences
chrisb at hgu.mrc.ac.uk
Thu Apr 11 04:31:03 EST 1996
Dr Alexander J Annala (ucklw08 at ucl.ac.uk) 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
: What alternative ways to grow and treat the bacterial culture should
: be considered in the instant situation?
I have often found that strains sick because of plasmid inserts grow
better at lower temperatures. Try 30 degrees, or even lower.
Remember also that in these circumstances there is strong selection for
cells containing plasmids that have deleted or otherwise mutated the
problem DNA. It is wise therefore not to propagate such strains
serially, but better always to seed from a stock frozen glycerol.
You could also consider subcloning the insert into a lower copy number
plasmid. Though this of course is unlikely to give you more DNA, the
strain might well grow more healthily as a consequence and make you less
concerned about deletions occurring.
Chris Boyd | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk | not | Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb | for | Edinburgh EH4 2XU, SCOTLAND
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