Northern Blots

[Jaishree Muppala Chittoor]

Help!  I am seeing a very intense background in all my blots, recently.  The 
hybridization protocol that I used was working properly (with no background) 
in all of my earlier blots. And now, all of a sudden, I am having this problem. 

I can't think of any parameter that I have knowing changed to account for the 
problem.  I have been using the same blot (magna, high density), probe, prehyb
solution (0.1% SDS, 0.05M phosphate buffer pH 7.0, 1M NaCl and 300u/ml salmon 
sperm DNA) with a prehyb time > 5hrs at 65 C in all my work.  I generally do 
hyb: overnight at 65 and washes with 2xSSC at 65. 

I changed blot (to gene screen plus), changed probe and even used a different 
pre hyb solution (5x SSC, 5x denhart, 0.5%SDS, 20mM phosphate buffer, 300u/ml 
salmon sperm DNA, 5mM EDTA), but nothing seems to eliminate the very intense 
background.  I have even tried washing with water.

I would greatly appreciate some help that will help me to solve this problem.

Bye
Jaishree [jaishre at matt.ksu.ksu.edu or jaishre at ksuvm.ksu.edu]

A
A
A
A