Uniform high backgroud in DIG Sounthern Blot - (Apr/26/2009 )
I have used the Roche Kit for my Southern Blot for 3 times, but every time, there was very high uniform backgroud (I used the Chemiluminescent Detection)!! I reduced the exposure time to as short as 20s, the backgound weakened, but the target signal weakened as well. I added hybridization buffer immediately after pouring out the prehybridization buffer, so I think the high background was not caused by the drying of the nylon membrane before hybridization.
I followed a detailed protocol based on the Roche Kit Manual from a senior labmate. He used a roller bottle (diameter similar to a cup) to perform the prehybridization and hybridization, but the volume of prehybridization buffer he used was only 10 ml which is suggested by the Manual to be at least 20 ml in the case of using a roller bottle rarther than a hybridizaiton bag.
Becuase he seemed to have got good results with the same protocol, I used 10 ml prehybridization buffer as well. But now I doubt if 10 ml buffer is insufficient to keep the membrane from drying during prehybridization in a such a big roller bottle........
Next time I would use 20ml, but I don't know if the problem would be solved this way.
Maybe the volume of prehybridizaiton buffer is not the key....
COULD ANYONE PLS SHARE SOME OPINION ON THIS??? THANKS A LOT FOR UR HELP!
try filtering the prehyb buffer with a .45um pvdf syringe filter before using. also, i would prefer a flat container for shaking instead a roller bottle. but then what works great for 1 person has to be re-optimised by another...also the prehyb can be reused many times. did you reheat the buffer to the hyb temp before using? also make sure when hybring or handling the prehyb buffer don't leave it for a prolonged time exposed to the air.
Thank you very much. My problem was solved months ago. And the reason for the extreme high backgroud seemed to be only because that the Blocking reagent which was offerred by a senior researcher in my lab was expired! Pity I had wasted so much time to repeat the experiment, only learnded that how hard it was to work with a stubborn senior guy as a freshman.