help needed with homogenizing

Nina Baltes nina at chaotropic.net
Fri Mar 21 04:02:16 EST 2003


Navinisha N Chetty wrote:

> I was wondering if anyone could help with a problem I am encountering 
> when trying to extract RNA from rat gut tissue.  At the moment I am 
> placing 30mg of gut tissue into lysis-binding solution (from the Ambion 
> RNAqueous-4PCR kit) and cutting it into tiny bits in an eppendorf tube, 
> with a pair of scissors, followed by homogenizing with a plastic pestle 
> provided in the kit.  I have found that I get very low yeilds of RNA 
> because the filter is always clogging during the isolation of RNA.  I 
> have also used the liquid nitrogen method with a mortar and pestle but 
> as I am using such small amounts of tissue, I loose much of the powder 
> when it melts on the mortar and pestle and thus again I get low yeilds 
> of RNA.  

Have you pre-cooled the mortar and pestle? The tissue shouldn't melt. 
The problem I've encountered with ceramic mortar & pestle is that the 
tissue powder adheres to the thing, frozen, and I can't get it out. I 
believe there are metal mortars that could help.

For extracting RNA from 30 mg porcine lung tissue (using the Qiagen 
RNeasy column), I've worked out the following which incidentally helps 
to relieve some aggression that may have accumulated ;): Take two metal 
plates (I'm using two metal lids for petri dishes that I found in the 
catacombs under our lab), freeze them in liquid nitrogen. Place one on 
the bench, put the frozen tissue onto it, cover with second plate, put 
some layers of paper on top so you don't make dents in it, and then give 
it a couple of good whacks with a hammer (be nice and warn your 
colleagues beforehand). Presto, lung powder. Then I use two frozen 
scalpel blades to scrape the powder into a tube containing the lysis 
buffer. No problem with clogged columns so far.

Nina
-- 
C'est les microbes qui auront le dernier mot.
                            Louis Pasteur
http://www.chaotropic.net



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