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PCR vs RT labeling with aa-dUTP

PCR vs RT labeling with aa-dUTP - Protocols and Methods Forum

PCR vs RT labeling with aa-dUTP - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 01-30-2006, 04:24 PM
Susan Hogarth
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Default PCR vs RT labeling with aa-dUTP



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
ask.

Thansk for any suggestions! As always, jsut writing things out has
cleared some things up for me and given me some thoughts.

- Susan

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  #2  
Old 01-31-2006, 03:09 PM
Duncan Clark
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Default PCR vs RT labeling with aa-dUTP

Historians believe that in newspost
<[Only registered users see links. ].net > on Mon, 30 Jan 2006,
Susan Hogarth <[Only registered users see links. ]> penned the following literary
masterpiece:

I take it you used straight Taq and not a proof-reading pol for the PCR?
Pfu and similar will not incorporate dUTP (or analogues) due to their
read-ahead function.

For Taq I would PCR using a dUTP:dTTP mix and verify that works before
using the more expensive aa-dUTP. Optimising the ratio is probably the
key, especially the longer the PCR product.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

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aadutp , labeling , pcr


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