USS DNA & mutagen. THANKS!!
jgraham at bronze.ucs.indiana.edu
Mon Jun 7 11:33:52 EST 1993
Good to see you have solved your problems. Actually low incorportation
of uridine into template DNA is not related to background problems
indicated by high transformation frequency of unprimed template samples.
This would be indicated by a low frequency of mutants from primed
samples with unprimed templates giving few transformants. You can test
the success of uridine incorporation by comparing transfection frequencies
on ung+ and ung- strains. However this is seldom a problem in my experience.
More important I think is clearing the phage(mid) culture of plasmid containg
cells, and removal of insoluble from the resuspended phage pellets, and
avoiding the release of small RNA's by RNAse treatments. In addition,
overgroing the superinfection culture will lead to cell death and lysis
and the release of polysomes precipitatable by PEG in collecting the phage.
As you point out ssDNA should not be released from the phage particle
until the DNA synthesis reaction is ready, as I see a nuclease activity
in many of these tempalte preparations, which can create short primers
from degrading templates on overnight storage.
Richardson Biochemistry Laboratory
Indiana University Bloomington
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