In vivo labeling of PUC19 Help!!!

Bernard Murray bernard at elsie.nci.nih.gov
Mon Jun 23 12:59:48 EST 1997


In article <33AD4CB7.1930 at pop.ben2.ucla.edu>, mleiblin at pop.ben2.ucla.edu 
says...
>
>Does anyone out there have a protocol for labeling PUC19 with =
>3H-Thymidine after electoporation into E. coli  so that the =
>resultant labelled plasmid can be isolated in a suitable state for =
>subsequent electroporations.  I would be interested in details such =
>as: 1) whether a minimal media is necessary to avoid the cold =
>thymidine in yeast based media; if so, which media and, 2) how much =
>labelled 3H-Thymidine in =B5Ci should be used to produce an "adequate" =
>specific activity so that 25 ng of labelled PUC19 can be detected in =
>a =DF counter.
>Any assistance would be greatly appreciated
>Thanks in advance
>Mickey Liebling

Just wondering....

Since plasmid purification is reasonably efficient, could you not
beef up the specific activity by adding a more general label (eg.
3H water) or any other precursor?  I suppose the opportunities
for radioactive contamination would be worse - don't autoclave
the medium!  :-)
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
(but truckin' to S.F. this week)




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