pcr

James M Wahlberg wahlberg at csd4.csd.uwm.edu
Sun May 23 20:17:26 EST 1993


Hello, molbio group.

	I'm interested in talking to people who use PCR to internally
biotinylate a DNA fragment (i.e., 1:2 biotin-dUTP:dTTP).  We followed
the protocol of Lo, et. al., from the Innis book, "PCR Protocols."
We have successfully produced a biotinylated 312bp fragment (S-blot
shows incorporation of biotin via Streptavidin-Alk. Phos conjugate).
So far, so good.  However, we have been unable to produce a larger
fragment (about 1kb) in the presence of biotin-dUTP.  The product is
amplified in the no-biotin-dUTP reaction, so we know the template and
primers and all else are OK.  

My questions are:

		1.  Anybody have any tips for amplifying larger fragments
		in the presence of this label?

		2.  Are there any special clean-ups we should try so we
		can digest our biotinylated product?  We've been unable
		to cut the 312bp fragment, even after CHCl3 and EtOH
		treatments.

So close, and yet so far!

			G'day!


			Jim   (wahlberg at csd4.csd.uwm.edu)



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