32-P labeling of PCR products.

[Shiao Y. Wang]

Zhongtang Yu wrote:
> 
> I would like to label the PCR products during PCR amplification
> to minimize any error which can be introduced after PCR reaction.
> Random labeling is not my choice for my quantitation of my PCR
> products by Phosphoimager.

To synthesize 32P-labeled DNA, we use 3 uM final conc of each of the
three cold dNTPs and 0.83 uM of the labeled nucleotide (5 ul of the 3000
Ci/mmol, 10 mCi/ml dATP from Amersham in a total reaction volume of 20
ul). Final primer conc is 0.25 uM. This is for high specific activity
probe. The rate of incorporation is pretty low (20-30%).

If you are interested in more efficient DNA synthesis, you need to
include a small amount of the corresponding unlabeled nucleotide and a
general increase in the nucleotide concentration. For example, Amersham
I think recommends 250 uM final conc for each of the three unlabeled
nucleotides, 50 uM (unlabeled) for the 4th nucleotide and 2.5 ul of the
labeled nucleotide in a final reaction volume of 50 ul. The optimal
molar ratio of cold/hot nucleotide needs to be determined empirically.

Good luck.

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