mike at medsun.unige.ch
Mon Mar 29 09:57:06 EST 1993
In article <1oaun9$9rb at gaia.ucs.orst.edu>, navarrer at ava.bcc.orst.edu (Roy Navarre) writes:
> 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!
1. Just to prove how much I hate the kit mentality (honest I do!), my
lab uses Quikhyb from Stratagene, for radioactive, single copy, human
Southerns, on 3-5 ug of DNA.
I find the signal good (I feel bad signals usually come from
inefficient transfers, but I would not swear to this) (only AT it). In
general, we have little bg - maybe some general "greying-out", but hardly
any spots etc.
2. We use a Stratagene "roller" oven - love it. All hybs in about 10 ml, and wash
in the same tubes. I only do up to two filters per cylinder, with nylon mesh
4. Stripping - we use NaOH (0.4M, I think; look at the recommendations with
the membrane). Oh, we use Boehringer's nylon, but have also used Hybond N
with no probs.
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