Use for Northern? (was ReRe: Non-radioactive labelling of DNA)

Arnoud van Vliet avvliet at knoware.nl
Fri May 5 16:41:41 EST 2000


Hi all,

that was a very interesting post about adaptations to the DIG protocols,
which I unfortunately missed previous time. Has anybody used this protocol
for Northern hybs? I am working with bacterial RNA, and DIG-labeled RNA
probes. Problem is that lots of probe stick to the 16S and 23S rRNA. Any
tips?

One of the things I wondered as well is whether one can autoclave the BSA /
Ficoll; I have to work RNAse free, so I prefer to be able to autoclave
stuff. Any hints on this one as well?

cheers
Arnoud



Susanne Rohrer <"srohrer(removethis)"@immv.unizh.ch> wrote in message
news:39119A15.D22F9D3E at immv.unizh.ch...
>
>
> "D. Durnford" wrote:
>
> > I would like to limit the use of 32P in my lab and I am shopping for
> > non-radioactive labelling methods (random prime). I am primarily
> > interested a system that is successful with Northern blot
> > Hybridizations. I would like to know if anyone would recommend a
> > particular system/method. I am concerned with stripping and
> > rehybridization, signal detection etc.
> >
> > Thanks in advance.
> >
>
> DIG labeling (Roche) works nicely: you can random label or use PCR
> probes, there is a kit for both. There is even one to chemically label
> probes-I haven't tried it yet. Southerns can be stripped, and with
> Notherns we haven't had great results but that's got to do with RNA, not
> the Dig system. Unfortunately it's not cheap. but then again you don't
> have the radioactive mess.
>
> there have been lots of posts discussing the method, and the standard
> one in the Roche DIG manual works.
>
> --
> Susanne Rohrer
> Institute of Medical Microbiology
> University of Zurich
> Switzerland
>
> srohrer at immv dot unizh dot ch
>
>
>






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