ss phagemid DNA

Anthony J. Persechini ajp2o at
Fri Nov 29 23:36:02 EST 1991

I recently started trying to use Stratagene's "Altered Sites"
kit for mutagenesis. I have cloned about 5 kb into their pUC
derived vector (~5 kb also). Attempts to isolate phagemid
DNA have been dismal.  I can barely see the
ssDNA bands when loading 1/4 of a prep from 1.2 ml
of phage supernatant.  The E. Coli strain is JM109.
I have tried growing the cells for 6 hrs or overnight
in 2XYT + tet after adding either R408 or M13KO7
helper phage. I have also tried adding 20 mM phosphate.
My last experience with ss phagemid preps was using pEMBL, with a much
smaller insert. Subsequent to this I worked with M13, also
with a small insert, and thought I was getting a low yield
THEN, at least by comparison with pEMBL.

Should I try growing the cells - tet? Perhaps
I should decrease the culture volume from 5 ml/50 ml tube
to 2ml/50 ml tube, in order to improve aeration?
I could scale this procedure up, but thought perhaps some useful alternative
suggestions might come of an inquiry to this group.

Anthony Persechini
Assistant Professor
University of Rochester School of Medicine
ajp2o at

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