Southerns/nylon membranes

Roy Navarre navarrer at
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!

More information about the Methods mailing list