32P into Supercoiled -No way ???

Karen KU at helix.nih.gov
Mon Nov 28 17:13:41 EST 1994


In article <mpowell-281194131500 at levinmac3.nichd.nih.gov>,
mpowell at lmgvax.nichd.nih.gov (Mike Powell) wrote:
> 
> 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-
> 

Thomas:

Do you really need the DNA supercoiled? If not then Mike's suggestion of
nick translation or something like random primer labeling would work fine.
If you really do need it to be supercoiled those options would not work. As
you suggested labeling the DNA in vivo is one way. Using orthophosphate
works well and is cheap but the plasmid isolation would involve a lot of
messing about with large amounts of radioactivity. A relatively simple
approach that works for smaller amounts of plasmid ( I dont know what you
consider "huge amounts"), would be to cut the plasmid with a restriction
enzyme that cuts once in the plasmid and generates a 5' overhang. P32 could
then be incorporated into the the terminal phosphates using T4
polynucleotide kinase, the plasmid molecule religated and supercoils
introduced into the molecule by treatment with Topo I in the presence of
ethidium bromide. If you need a protocol I can send you one.

Karen

******************************************

Karen Usdin
Section on Genomic Structure and Function
NIDDK, National Institutes of Health

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