Function of dUTP in real-time PCR?
tk at csail.mit.edu
Fri Oct 29 10:10:43 EST 2004
paul_wary at yahoo.com (Paul Wary) writes:
> Ummm, where do I actually get any carryover PCR product from? I use
> real-time PCR for quantification of gene expression levels, so I
> reverse transcribe mRNA to cDNA and use that cDNA for the real-time
> PCR. I can't see where I get that carryover PCR product from.
Well, that's the point. You're *not* supposed to get carryover. But,
because in principle PCR can amplify a single copy of a gene, it is
difficult keep reagents, benchtops, pipets, and air free of copies of
the gene you are interested in. Yesterday you amplified 10**15 copies
of that gene. Some probably ended up on your bench. Some of those
probably ended up in the tube you are PCRing today. You would like
those not to be templates.
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