Jaishree Muppala Chittoor
jaishre at matt.ksu.ksu.edu
Sun Nov 8 23:01:18 EST 1992
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.
Jaishree [jaishre at matt.ksu.ksu.edu or jaishre at ksuvm.ksu.edu]
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