oligo dT column too slow

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Wed Jan 21 11:32:00 EST 1998


At 00:07 1/21/98 -0600, Kent S. Boles wrote:
>Hi all,
>I just made an oligo dT cellulose column in a 3 ml syringe barrel with 100
>mg of cellulose plugged by glass wool.  I'm also using Sambrook's protocol
>and binding and elution buffers.  After loading my sample of total RNA to
>the column, I washed with about 20 mls of binding buffer and still had RNA
>in the effluent.  The column had become incredibly slow with salt crystals
>forming in the cellulose.  The room temp was about 23 degrees.  I gave up
>and eluted my sample in excess elution buffer.  It still contained clearly
>defined ribosomal bands.
>I've only  seen some vague references as to the construction of these
>columns and companies only want to sell me the prefabs.  Does anyone have
>some specific suggestions to increase the efficiency of this process or some
>observations on mistakes that I might be making?

Kent:

I think I can help.  I get my Oligo dT columns from Collaborative Research,
which if memory serves is distributed by BD.  I got them through the
"Fischer" middle man.

1)  Not all of that cellulose hydrates.  Before adding your precious RNA,
establish the flow rate with stripping buffer and binding buffer.  Stir the
column with (of course!) a baked, RNAse-free glass pipette or a single use
sterile plastic one.  A lot of the un-hydrated globs of cellulose end up at
the top and need to be removed to establish a good flow.

2) and most important, substitue LiCl for NaCl in the Maniatis protocol.
Yes, that may mean remaking the gaggle of the solutions you just
pain-stakingly made, but would you rather deal with the SDS coming out of
solution?

I've tried the Invitrogen fast track and it worked, for the most part.  I
found it to isolate quality mRNA from tissue culture cells fairly well.
However, for some reason, I could not get decent mRNA from stimulated HL-60
or U937 cells.  It almost always came out degraded.  Also, the overall
yeild is low, albeit fairly pure when it worked for other cells.  The most
I've ever obtained has been about 5-7 ug per their protocol.

With the big column, I've obtained anywhere from 20-100 ug PA+ mRNA, from
tested batches of total RNA that likely amount to about mid-10^9-10^10
total cells, or anywhere from 3-5 mgs total starting material.  

Yes, with the column that big, and that much total RNA, I can usually plan
on running the column for 6-7 hours.

Hope this helps,
David

=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



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