Northern Blot - RNase-free

[GysdeJongh]

Hi Simone Marker,

"Simone Marker" <marker from rhrk.uni-kl.de> wrote in message 
news:f8n6cj$st8$1 from news.uni-kl.de...
> Hi,
>
> In general, when I prepare a denaturing agarose gel (with formaldehyde) 
> for separation of RNA do I have to make the solutions and the apparatus 
> RNase-free?

No , the formaldehyde in the denaturing buffer will stretch your RNA so that 
a precise determination of their molecular weight distrubution is possible 
.But ....... it will also denature the RNAses ; they are just proteins after 
all.

> Is it the same for denaturing polyacrylamid gels with urea as denaturing 
> reagent? (I want to detect small RNA (siRNA) by northern)

Yes and / or the guanidine salt or other chaotropics

> And when I continue with blotting for a northern, which steps need to be 
> RNase-free?

Think about it :
the purpose of that type of experiment is solely to determine the molecular 
weight distribution of your RNA molecules.So after the gel elektrophoresis 
the RNA molecules are on the right spot to make just that possible.The only 
thing left is : you need to make them visible.So if during the blotting 
process or during the detection the RNA is degraded it will still be on the 
right spot and the degraded molecules will still react with your radioactive 
probe.So after the electrophoresis any precaution for RNAses is not 
necessary  :)

>Is it really necessary to prepare RNase-free Whatman-paper,

No

> membrane, vacuum blotter and so on?

No

>What is the easiest way to do this? I

Don't do it

> heard of wiping the equipment with H2O2, but I'm not sure about the 
> concentration. And how can I get Whatman paper RNase-free?
> Or do you think that working quickly might suffice to reduce degradation 
> by RNases?

Working quickly is not for good scientists ; they are smart and lazy  :)

hth
Gys