Southern Blotting - Mystery in southern blotting (Aug/07/2009 )

I faced some problem in my southern blot. Instead of black bands, I am getting white bands. If there is anyone who can explain the problem and how to fix it would be appreciated.

dawitgirma1@yahoo.com on Aug 7 2009, 08:23 AM said:

it sounds like your probe concentration is too high or the detection method is too strong and causing negative staining....do the bands look sort of like a halo on the film...clear in the centre with a faint dark ring around the signal?

are you using HRP? if yes, then you need to dilute it.

eldon on Aug 7 2009, 11:04 AM said:

thank you so much for your explanation. well i think i did the detection step more that for 5 minute and that might be one of the problem. the band looks like just like you said. i used DIG probe method for detection.
The other problem on my southern is that, i am getting band on lanes of the membrane which do not have any sample (from empty wells). what is the cause of that problem too?

just use less secondary antibody with the DIG system...or alternatively, if you have the problem again just wait 30 min. or so and the signal should begin to fade.

you might be overloading the wells and getting spill over...concentrate your samples and load less or try dry loading if you can't load less. make sure your blot stack isn't moving during transfer etc., although if this happens it's pretty obvious..it looks like accidental re-positioning of film on your membrane when the signal is strong.