PCR to make small labeled probes

Brian Foley brianf at med.uvm.edu
Tue Feb 1 09:39:31 EST 1994


S. Gupta (Sugupta at ccmail.sunysb.edu) wrote:
: Has anyone tried to make a 32P labeled DNA probe to do a library screen? 
: The size I am trying to amplify will be around 100bp.  Any suggestions
: will be appreciated.

	Works great!  Just use 1/10 the normal concentation of the 
dNTP that you will use as label (for example if you will use alpha-
32-P-dCTP then use 20 micromolar cold dCTP when you would normally use
200 micromolar, keep the other dNTPs at 200 micromolar).  Then add
the labelled dCTP.  The molarity of the dCTP (labelled plus cold)
will vary depending on the activity of your labelled dCTP, but this
does not present a problem. 
	Keep the number of cycles low, like 10 cycles and use a 
relatively pure template (i.e. plasmid or previously amplified 
PCR product) rather than genomic DNA.
	Run a very small aliquot of the product on a gel if you
want to check that the product is full-length and contains
no artifacts.  Purify the incorporated label from unincorporated
free dCTP by the same method you normally use.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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