Fundamental Northern Blot question

"JEJS Jan Eric Jensen JEJS at NOVO.dk
Mon Jan 24 04:39:01 EST 2000


Hi,

As to reduce signals from stains and/or high background you might try out
this procedure.

I treat the still moist/wet membrane as follows:

Soak a Kimnet paper towel in 65 degC 0.1xSSC/0.5% SDS.

Place the wet membrane on the soaked paper towel.

Soak another paper towel in the warm SCS/SDS-solution and use it to wipe
both sides of the membrane (a great deal of force can be used without
reducing specific hybridization signals).

Wrap the menbrane in Saranwrap and the membrane is ready for
autoradiography.

I've only tried this on Hybond-N, so it might not be workable on more
fragile membrane types.

Good luck.

Regards,

Jan




 

> -----Original Message-----
> From: Frank O. Fackelmayer [mailto:Frank.Fackelmayer at uni-konstanz.de]
> Sent: 24. januar 2000 09:53
> To: methods at hgmp.mrc.ac.uk
> Subject: Re: Fundamental Northern Blot question
> 
> 
> 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
> POST!!!).
> 
> Good luck,
> Frank
> 
> 
> 
> 
---




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