32P into Supercoiled -No way ???

Mike Powell mpowell at lmgvax.nichd.nih.gov
Mon Nov 28 13:15:00 EST 1994


In article <3bcqiv$lpc at infosrv.edvz.univie.ac.at>, Thomas Hiesberger
<hiesi at mol.univie.ac.at> wrote:

> Hello
> 
> In one of our experiments it would be necessary to label circular,
> supercoiled plasmid DNA with 32 P. At first, it did not look difficult at
> all. But now I am kind of desperate, because I can not think of a proper
> way to do this, apart from making single strand DNA and synthesizing the
> second strand using 32P nucleotides - I need huge amounts and do not get
> it with this method.  The other possibility is to grow bacteria harboring
> the plasmid in a medium containing 32P - The 32 P incorporation
> efficiency seems not to be very high (32P would also incorporate into
> genomic DNA and using chloramphenicol for amplification does probably not
> go well together with 32P so I do not really want to go into this). But I
> still think there must be a way of labeling I have not thought about. I
> would be very grateful for any idea or advice. 
> 
> 
> Thomas Hiesberger

Thomas,

  Maybe you could try "Nick Translation" to label the plasmid
DNA?  There are several protocols but the one in Maniatis book
two page 10.6 might give you a starting point.

-mike-

*********************************************
When the going gets tough, I fall behind.
*********************************************
Mike Powell, PhD
Laboratory of Molecular Genetics
National Institutes of Health
Bldg 6B  Rm 216
Ph: 301 496 5538  Fax: 301 496 0243
email: mpowell at lmgvax.nichd.nih.gov
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