ss phagemid DNA

Anthony J. Persechini ajp2o at crocus.medicine.rochester.edu
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 crocus.medicine.rochester.edu



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