Terrence P. Delaney
tpd4 at cornell.edu
Thu Apr 17 18:31:33 EST 1997
The protocol below (by Michael Tuvim) uses much more radioactivity than
needed and seems kinda' involved. Another protocol posted (by Chi-Kuang
Wen) recommends 1ul of hot stuff (3000 Ci/mM) in an 11 ul rxn; this will
produce a reaction mix weel BELOW the kM for TAQ. My compromise follows:
To produce quality high specific activity PCR probes, I dry down approx 15
ul cCTP (3000 Ci/mM) in a speedvac (place a punctured cut-off eppy cap
onto the eppy you'll do your PCR in, and place both in the speedvac for ca
15 minutes to dry; monitor afterward for contamin'!).
Prepare the mix of your buffer, primers, substrate, nuc's (minus dCTP)
plus TAQ, and add 10 ul of that onto the dried hot stuff.
Do about 15 cycles of PCR and you should have a probe with 60-70%
incoprporation, and exremely HOT.
The values for the aforementioned reagents are as you would use in any
PCR; just scaled down. By concentrating the hot stuff down about 2/3, the
kM nuc's for TAQ is approximately achieved.
Terrence P. Delaney, tpd4 at cornell.edu (607) 255-7856 (fax-4471)
Cornell University, Department of Plant Pathology
341 Plant Science Bldg, Ithaca, NY 14853
In article <5j35i4$2qp at gazette.bcm.tmc.edu>, mtuvin at bcm.tmc.edu (Michael
J. Tuvim) wrote:
> In article <Pine.SCO.3.91.970415211117.14284D-100000 at agatha.unr.edu.ar>
jirazoq at AGATHA.UNR.EDU.AR (Javier Irazoqui) writes:
> >How should I radioactively label DNA probes by PCR, obtaining a high
> >specific activity?
> Here is my protocol. It works really fine.
> SYNTHESIS OF UNIFORMLY RADIO-LABELED DNA PROBE BY
> "ARITHMETIC" POLYMERIZATION
> Careful: this protocol requires a lot of HOT STUFF.
> Note: you may cut everything by half if you don't need all that much.
> Amersham sells nucleotide either by 25 ul or 100 ul, and the latter will
> cost you.
> 1. Take 50 ul (500uCi, 3000 Ci/mmole, 10 uCi/ul) of alpha-P32-dCTP (Amersham
> #PB10205 or equivalent - ain't havin' any dyes or nothin'). That
> gives you 160 pmoles of the nucleotide. Dry it down completely in a
> speedvac at RT overnight. Run parallel dummy: 2.5 ul of
> 2-mercaptoethanol in 50 ul of water. Check if 2-mercaptoethanol is
> still there in the morning: sniff the dummy.
> 2. Redissolve in some (say 20 ul) water (DEPC-treated if to use with the
> 3. Add 2 ul of 5 mM solution of each cold dATP, dTTP, dGTP (gives you
> 10 nmoles each).
> 4. Add 10 ng of your template and whatever amount neccessary of your
> primer to get 2 uM.
> 5. Add 5 ul of 10x PCR buffer.
> 6. Add 5 ul of MgCl2 25mM soln.
> 7. Add water to 50 ul.
> 8. Add 0.5 ul of Taq Polymeraze (we use Promega's enzyme and buffer).
> 9. Cycle 20 times at: 94C for 1', 40C (or whatever you see fit for your
> primer) for 1', 72C for 1', no final extension, leave at 4C.
> 10. Put through gel filtration column (we use Bio-Rad Bio-Spin 30 or
> Pharmacia Nick-Spin), repeat if you got a lot of noprecipitable
> counts (we haven't).
> 11. Heat it up to 94C, add salmon sperm DNA, chill on ice. [This step is
> optional since you really you don't need to worry about renaturation
> of your probe.]
> 12. Enjoy your probe.
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