Southern Blot problem - (Oct/19/2006 )

Hi, I've been doing southern blots of a gene for awhile with beautiful strong bands after exposing to film. After I ran out of the probe, I made some and can no longer get the same results. I used the same primers, made fresh primer stocks, had the probe sequenced and tried a few concentrations of DNA in making the probe but it still produces weak bands and now some background.

I use stratagene quikhyb, p32, hyb for 1.5 hours at 65C, 1st wash: 2X ssc for 15 minutes, 2nd wash 0.2 ssc/0.1 SDS for 30 minutes-both at 65C. This produces always for me perfectly clean films and nice bands.

Anyone have a suggestion? Thanks

Could it be that the labeling of your oligo goes wrong? are you using same conditions as with the one that was working for this?