help needed with homogenizing

Michael L. Sullivan mlsulliv at wisc.edu
Mon Mar 24 09:53:11 EST 2003


Something we use for small amounts of plant tissue is to use a bead 
beater to powder the tissue in liquid nitrogen.  We place the tissue 
in a 2 ml screwcap tub with two 4 mm glass beads.  We place the tube 
in liquid nitrogen to freeze the tissue (a styrofoam rack for 
disposable cuvettes works well for this).  We then use the bead 
beater at full speed for 5 second pulses.  Between pulses, we place 
the tube back in liquid nitrogen to keep it frizen.  Hope this helps.

Mike

>Hi there!
>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.  The gut tissue is very mucousy and stringy and I do not know
>of any other methods to obtain a high yeild of RNA from a small amount
>of tissue.  Can anybody please help me???      
>Thanks,
>Nav
>
>---


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Michael L. Sullivan, Ph.D
Research Plant Molecular 
Geneticist                                                            
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax
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