PCR labeling

Chi-kuang Wen 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)
primer1                 1ul(50nmole)
primer2                 1ul(50nmole)
10XPCR buffer           1ul
Taq                     1ul
50uM dNTPs(-dCTP)       1ul
P32-dCTP, 3000Ci/mmole  5ul

total 11ul

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.

Chi-Kuang Wen

Botany & Plant Pathology
Purdue University
W Lafayette, IN 47907
 





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