ckwen at expert.cc.purdue.edu
Wed Apr 16 16:09:36 EST 1997
Michael J. Tuvim (mtuvin at bcm.tmc.edu) 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.
Here is a protocol requies a small amount of hot stuff.
DNA template in vector: 1ul(1ng)
10XPCR buffer 1ul
50uM dNTPs(-dCTP) 1ul
P32-dCTP, 3000Ci/mmole 5ul
Go to your PCR cycles
Remove oil, add 80ul TE, pass through a 3~4ml of SephadexG100 by spinning
at 1000rpm for 3~5min
Collect the eluted solution and boil for 5min, chill on ice and ready to go.
Botany & Plant Pathology
W Lafayette, IN 47907
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