Northern blot and NybondN+membrane

ruth e.w. walker pharmrew at muccmail.missouri.edu
Fri Mar 8 14:14:02 EST 1996


I am very happy with Nylon membranes (use BioRad Zetaprobe) and have 
seldom had problems with background. When I have, it has been due to 
(sometimes both, as they are not mutually exclusive) either

1. Not adequately denaturing the probe. If the probe is not thoroughly 
denature I will see hot pinpricks all over the autorad. Nothing to be 
done for this except strip the membrane and rehybridize; or

2. Not washing stringently enough, which for me means just not long 
enough. If there appears to be generalized (not punctate) background, I 
will use the stringency wash again. (I hybridize with Clontech's 
Expresshyb at 68C for 2 hr, rinse 4 times briefly with room temp 2x SSC, 
then wash with 0.05x SSC/0.1% SDS twice for 20 min).

Honestly, they look clean as a whistle.  




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