DIG Southern Blots - Signal but very high background (Jun/03/2005 )
I am doing DIG southern blotting on plant gDNA using a variety of DIG labelled PCR probes that have been amplified from cloned products. I am getting signals in my blots but I'm having real problems with background. In some cases its so bad it obscures the signal!
I've tried to reduce the background in my Southerns by following the Roche guidelines but so far with no success. Does anyone have any suggestions on how to achieve these lovely background free southerns that I keep seeing pictures of?
This may sound basic but are you sure you've got your chemiluminesence detector on the right settings? I kept getting horrible background until I found the noise filter on our Aplha Innotech machine.
I also do some extra detection steps recommended to me by Roche technical support:
Incubate membrane for up to 2 hours in blocking solution, rather than 30 min;
Do an extra 15 min incubation in washing buffer, and use up to 250 ml of washing buffer;
Give a quick rinse in detection buffer before the 5 min incubation in it.