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Fundamental Northern Blot question

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Jan 24 03:53:01 EST 2000

Karthik Aghoram wrote:

> Hi,
> I have a couple of quick questions about Northern blotting.  I use
> GeneScreen Nylon membranes to capillary-blot polyA RNA.
> Hyb with gene-probe1 -- 50 degC, wash 3-4 times at hyb temp; nice clean
> blots.
> Strip blot by boiling in 0.1 X SSPE; 1 % SDS for 15-20 min.  Take care to
> avoid drying; creases etc.
> Hyb with gene-probe2 -- same conditions as first hyb.
> Result:  Blot covered with ugly spots.  Even high-stringency washes don't
> work.  Heck, one time reboiling didn't take it off.
> Haven't tried hybing with probe 2 first.  Will try.  But any of y'all have
> any ideas why this may be?
> Thanks a lot.
> Peace.

Hi Karthik,
Ugly spots on Northern are often due to:
1. Powdered gloves. NEVER use powdered gloves in molecular biology work. The
powder can easily ruin your work when it gets into your solutions or vessels.

2. unincorporated nucleotides in the probe. Remove unincorporated NTP by gel
filtration (eg. over nick-columns or homemade Sephadex G50 columns)
3. particles in the hyb solution. Filter hyb solution before use.

Once on the membrane, the ugly spots cannot be removed again, and the
membrane is usually ruined (IF ANYONE HAS A PROTOCOL TO GET THEM OFF, PLEASE

Good luck,

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