Northern - Cross Hybridization Problems (Jun/01/2002 )
I am using Ambions NorthGly Kits and as a control probe using Bactin that is nonisotopically labeled with biotin (also their kit), hybridizing at 68C. On my blots I am getting a strong rRNA hybridization. I am using the kits washing buffers which are 2XSSC/0.1%SDS and 0.1XSSC/0.1%SDS at 68C. I have on my blots 30ug total RNA that was extracted from 293 cells and control RNA from kit.Are there any suggestions to eliminate this problem? I am using my probe at 1pmol/10ml for 100cm2 blot. I have cut the probe conc. by 50% and still seeing some cross hybrid. I welcome any ideas!
Let's try raise the SDS % from 0.1 to 1% in the washing buffer