Background problem with a northern blot

William Driscoll driscoll at rci.rutgers.edu
Thu Aug 14 16:42:50 EST 1997


I am having a background problem with my northern blots.  I am getting an 
large amount of non-specific binding.  I am using a biotin labled 40mer 
DNA probe.  My membrane is a nylon positively charged membrane(Boehringer 
mannheim).  We have narrowed the problem down to the hybridization 
blocking step. The hybridization conditions are the following: \
	Temp: 65 degrees C 
	Solution:7% SDS, 37g/L EDTA, 35.5g/L Sodium phosphate dibasic

I would appreciate and help or advice about what to do to reduce this 
non-specificity problem.  I was wondering what others have added to 
the reduce the background when using a biotin labled probe.  I was thinking 
of adding herring sperm DNA but I don't know where to obtain this. (I've 
tried pharmacia, sigma, Fisher, and BioRad.) I also was thinking of 
adding Denhardt's Solution. 

I don't get to check news as often as I'd like so please email me (driscoll at rciu.rutgers.edu) instead of just posting (I might miss the post) ... Thank you

Thanks in advance

Bill
driscoll at rci.rutgers.edu 



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