Southern Blot problems - Help needed to fix southern problem!! (Oct/05/2006 )

Hi,

I have completed many many southern blots but recently I can't make any of them work and i need to find out how i can fix it quickly, the work is urgent! I am getting a very high background level (i need it to be clean to quantify but you can't even see some bands because of background level on some of these gels). I have tried changing things but the background is only getting worse.

The agarose gels are fine and very clean. I blot using skimmed milk and dextran sulphate with 2 x SSPE and 1% SDS. I tried removing the dextran, it slightly improved but still filthy near edge of membrane and had dots over it. Increasing SDS to 2% and making new SSPE didn't help either, nor did using 10x denharts for hybridisation instead of dextran. Could it be the P32?

The background is usually big smears across the membrane (as opposed to spots) but sometimes also get big intense spots. It seems to not be blocking properly i think, very weird. I am leaving it to pre hyb overnight but this doesn't help

Any suggestions?

Thank you so much in advance for your helpful suggestions! Its driving me crazy!!!

I don't know what it is, but you can debug it more easily if you skip the gel, skip the transfer, and start with spotting the probe on the membrane in a dilution series and make sure that you can detect that. Then, you spot the raw sample in dilution series on the membrane, and make sure you can cleanly block, hybridize and detect that. Only then do you bother to run gels. In other words, work backwards and make sure everything is working at the end, then start at the beginning.