Weird Northern again!!!

SURESH KUMAR K.G. skg at biochem.iisc.ernet.in
Tue Jul 25 10:29:52 EST 2000


Hi,

Are you by any chance overloading your gel with the RNA....?
If your gene of interest is expressed at a fairly decent level
you may try to load less amount of total RNA and also as some one has
rightly pointed out earlier,use a higher starting temperature for hyb.

Hope this helps...

Suresh Kumar K.G.
Dept of Biochemistry
IISc, Bangalore-12 , India.

On 24 Jul 2000, Chunxin Wang wrote:

> The oven is ok as I put a thermometer inside as a control. I also tried
> two different hybridization buffers, one is the standard (Denhardt's, SDS
> and SSC), another is the Church's buffer (7%SDS and NaPO4 buffer) and the=
y
> produced the same result. One thing now came up to my mind is that the
> three probes hybridized to the rRNA are all from PCR amplification, while
> the only probe worked well so far is from a EST clone. Does this account
> the difference? Also, is it possible that the labeling kit gets
> contaminated by unkown reason?
>=20
> Black
>=20
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> On Mon, 24 Jul 2000, Frank O. Fackelmayer wrote:
>=20
> > Hi Black,
> > We=B4ve had some hybridization problems years back. We checked everythi=
ng.
> > Finally it was the oven that did not keep the set temperature but was
> > much colder and gave unspecific hybridization.=20
> > Check it. If the oven and you buffers are correct, try a control hyb
> > with an abundant message.
> >=20
> > Frank
> >=20
> >=20
> > Chunxin Wang wrote:
> > >=20
> > > This time two new probes PCR-amplified from arabidopsis PR-1 and VSP1
> > > genes, repectively, were used and the membrane was newly transferred.=
 I
> > > did the hybridization at the same time in different bags. However, th=
e
> > > result was the same: all signals came from the rRNAs. The hybridizati=
on
> > > pattern was exactly the same as the EB-stained gel pattern.
> > >=20
> > > why it happened to me so often in so short time?I was so desperate no=
w.
> > >=20
> > > Black
> > >=20
> >=20
> >=20
> > > ---
> >=20
> >=20
> >=20
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> ---
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