Southerns/nylon membranes

Roy Navarre navarrer at ava.bcc.orst.edu
Thu Mar 18 17:56:41 EST 1993


In the past I have used nytran and prehybed and hyb'd in 6x SSC and 1% SDS,
no denhards or Salmon sperm DNA. I never had any problem with background.
Lately, I have started doing southerns in a glass tube, in a rotating
hybridization oven. I have switched over to zetaprobe and .25M NaPO4 and
7% SDS. Results have been generally very bad. Pinhead type spotting is
a bad problem. Probe is diluted before adding it to the hyb solution.
I am concerned that the high SDS concentration is causing a precipitant to
form, and this is the origin of the spotting. Furthermore, membranes cannot
be stripped clean afterwards. I have tried everything from mild washes, to
boiling in SDS. Anything strong enough to remove the non-specific background
also removes the sample DNA.

Can anyone share their experiences of the following with me? :

1. 7 % SDS hyb and prehyb solutions. Do you get spotting?

2. Hybridization ovens. Satisfied with the results? Pitfalls?

3. Different nylon membranes. Are some less suited for 7% SDS solutions?

4. Stripping nylon membranes. We work with single copy plant genes. Losing
10-20 percent of our signal intensity with each stripping cycle. Less harsh
procedures dont remove background. Can anything be done about this?

Any help greatly appreciated!
Thanks
Roy



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