PCR with 32P-dCTP?

Shiao Y. Wang sywang at whale.st.usm.edu
Fri Nov 21 13:48:04 EST 1997


Bill Geese wrote:
> 
> FOr a "typical" 50ul reaction (2mM MgCl2, 200uM dNTPs, 20pmol each primer,
> 1ng plasmid template) when I use 32P-dCTp I add anywhere btw 10-50uCi
> (depending on the purpose) and cut the cold dCTP to 100uM.

Hello Bill. The problem we see is that if you add 50 uCi, the
concentration of hot dCTP is 0.33 uM. If one adds 100 uM of cold dCTP,
the specific activity of the probe (assuming one is trying to synthesize
DNA probes) becomes many times lower than that produced by random
priming. I suppose that's why many companies are still selling random
priming kits. I would be very interested to know if anyone has worked
out the best ratio of hot/cold nucleotide to use for probe synthesis and
the specific activity of the resulting probe.

-- 
Shiao Y. Wang
University of Southern Mississippi
sywang at whale.st.usm.edu



More information about the Methods mailing list