Chef gel blotting problem

Mark S. Rose msr2 at cornell.edu
Fri Feb 4 18:54:17 EST 1994


Hi folks
Some people in our lab have been experiencing problems with blots of
chromosomal  preparations run on CHEF gels.  Visible amounts of DNA in the
form of reasonably well resolved chromosomes are present in our CHEF gels. 
However when these gels are blotted to Hybond membranes and probed no
signal is visible despite long exposure times (~1 week), use of very hot
probes, and apparent absence of DNA in the gels after the transfer
procedure.  For these blots we've been using our usual transfer procedure
(1/2 hour in 0.2 N HCl, rince briefly in H2O, soak twice for 20 minutes
each in 0.4M NaOH, 1M NaCl, transfer in 0.4M NaOH, 1M NaCl overnight, rinse
blot briefly in H20, dry and bake).  Any suggestions would be appreciated.
Thanks
Mark



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