USS DNA & mutagen. THANKS!!

dnicker at vax.oxford.ac.uk dnicker at vax.oxford.ac.uk
Mon Jun 7 14:30:58 EST 1993


In article <C89FCG.Azv at usenet.ucs.indiana.edu>, jgraham at bronze.ucs.indiana.edu
 writes:

> Darren,
> 
> 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.


Jim,                                      

I did not mean to imply that low uracil incorporation was related to our
observation of plaques on the control mutagenesis (USS template but no primer). 
The fact that these plaques were due to self-priming as a result of poor
quality template (contaminated by short fragments of DNA with the ability to
prime) was meant to be a separate point from the above 2 which discuss poor
uracil incorporation.
As well, your experience has been more pleasant than my own.  We did in fact
have a problem with uridine incorporation, which we now realize was due to an
overly high MOI.  The phage was titred on CJ236/MV1190 and transfection
frequencies were only 100-fold less on MV1190.  In order to expect an
appreciable mutagenesis efficiency I am told this must be at least a 10^4 or
10^5 fold reduction.  I plan to re-infect with this unacceptable U-phage
tomorrow in CJ236 which should solve the problem, and I will re-titre.
As regards your other comments on clearing the phage culture of various cell
remnants and avoiding lysis by overgrowth, this is indeed valuable advice.  It
has been echoed by others and I was remiss in not including it in my summation.


							Darren Nickerson
						Inorganic Chemistry Laboratory
							Univ. of Oxford



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