Northern Blots

Martin Kennedy cytogen at chmeds.ac.nz
Mon Nov 9 23:00:40 EST 1992


In article <1dknqeINNt6r at matt.ksu.ksu.edu>, jaishre at matt.ksu.ksu.edu (Jaishree Muppala Chittoor) writes:
> Help!  I am seeing a very intense background in all my blots, recently.  The 

....stuff deleted...
> 
> 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 find GeneScreen Plus requires higher concentrations of salmon sperm DNA for
blocking than several other membranes.  If the black background doesn't wash
off then it's covalently bound to your membrane, and the membrane probably
wasn't sufficiently blocked in the first place.  Try using ca 500ug/ml ssDNA in
your prehyb mix; I also add a further 1-2mg of ssDNA to my probe when I boil
it, and then add about 10ml of hyb mix before tipping it into the bag/bottle.
As for the black membranes, put them away for a few months!

> Jaishree [jaishre at matt.ksu.ksu.edu or jaishre at ksuvm.ksu.edu]
> 
Good luck!

Martin



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