Fundamental Northern Blot question

Karthik Aghoram kaghoram at garnet.acns.fsu.edu
Sun Jan 23 17:02:16 EST 2000


Hi,

I have a couple of quick questions about Northern blotting.  I use
GeneScreen Nylon membranes to capillary-blot polyA RNA.  

Hyb with gene-probe1 -- 50 degC, wash 3-4 times at hyb temp; nice clean
blots.

Strip blot by boiling in 0.1 X SSPE; 1 % SDS for 15-20 min.  Take care to
avoid drying; creases etc.

Hyb with gene-probe2 -- same conditions as first hyb.  

Result:  Blot covered with ugly spots.  Even high-stringency washes don't
work.  Heck, one time reboiling didn't take it off.

Haven't tried hybing with probe 2 first.  Will try.  But any of y'all have
any ideas why this may be?

Thanks a lot.

Peace.




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