PCR vs RT labeling with aa-dUTP
hogarth at gmail.com
Mon Jan 30 11:24:07 EST 2006
I'm looking for suggestions on PCR labeling with aa-dUTP.
I have done aa-dUTP (followed by Cy3/5 coupling) using RT with
amplified RNA and had no problems. Now I am trying to label PCR
products the same way, and the rate of incorporation is *much* less,
although I seem to be getting a decent amount of the PCR product.
An additional wrinkle is that I am doing asymmetric PCR so I get an
excess of ssDNA.
Thoughts? Anyone care to share PCR labeling protocols? Most of what I
find is centered around RT labeling, although Jena Bioscience has a PCR
labeling kit whose protocol I am basically following. The Jena protocol
uses a aa-dUTP:dTTP ratio of 1:3 rather than 2:3 which was used in my
RT protocol. I think that will be my first thing to vary - upping the
aa-dUTP:dTTP ratio to 2:3. There is a note with the Jena protocol (and
I seem to have read it elsewhere) that "depending on DNA template
incorporation efficiency may decrease with increasing fragment
length"). I have several fragments, but the one I am using for testing
is about 1kb (it's probably my longest probe).
I do have one concern - my last reactions stayed over the weekend at 4c
instead of -20c after the pCR reaction and before the Cy-dye coupling.
Does storage at 4c affect the aa-dUTP badly? I am goignt o go ahead and
clean it up and do the coupling reaction regardless, but I thought I'd
Thansk for any suggestions! As always, jsut writing things out has
cleared some things up for me and given me some thoughts.
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