ss phagemid DNA
num208ww at MBDS.NRC.CA
Sun Dec 1 22:35:08 EST 1991
In article <1991Nov30.043602.6231 at galileo.cc.rochester.edu>, ajp2o at crocus.medicine.rochester.edu (Anthony J. Persechini) writes:
>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.
>University of Rochester School of Medicine
>ajp2o at crocus.medicine.rochester.edu
I have had quite good success with phagemids as long as your helper
phage is good and healthy. A problem with low yields may be due a low
titre on your helper phage. A second potential problem is your strain
of E. coli. JM109 is sick, and doesn't respond well to phage
infections especially if a plasmid is there. It tends to lyse easily.
Try one of the similar but less cripled versions like MV1190 or TG1.
Dr. Warren Wakarchuk, Internet: Wakarchu at biologysx.lan.nrc.ca
Protein Structure & Design, or NUM208WW at mbds.nrc.ca
Institute for Biological Sciences,
National Research Council of Canada.
==> These opinions are MINE, not NRCs !! <==
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