Problems with Southern Blot - Probe labeling - (Aug/22/2007 )

Hi there.

These forums are great! There is so much information here.

I am hoping someone here may have an answer for me. I am having trouble with my Southern and I think I have it narrowed down to a problem with labeling the probe. I am using Invitrogens "Random Primers DNA Labeling System," and 32P is my radioisotope. Since 32P has a short half-life (~14 days) I have been doubling the amount of 32P added to compensate for the decay.

Should I also bump up the amount of the other reagents I am using? Or should I only use 32P that has not decayed at all?

Any comments would be greatly appreciated. Thanks!

i assume that you are using gamma-32p-atp to label. if so, then by doubling you will be increasing adp-s (after decay of 32p) in the reaction.

your best bet is to use freshest available in the amount recommended (without "doubling") and start with the highest specific activity you can.