Can the oligodT coloum be reused?

Andre Hamel hamel at ccu.umanitoba.ca
Tue Feb 16 15:11:24 EST 1993


In article <1lfdo3INN8nc at matt.ksu.ksu.edu> jaishre at matt.ksu.ksu.edu (Jaishree Muppala Chittoor) writes:
>I would like to know whether oligodT cellulose column can be reused for mRNA 
>purification.
>Jaishree
>[jaishre at matt.ksu.ksu.edu]
>
Just wash the oligo dT cellulose in 0.1 N NaOH (if in a column wash the
column with about 5 column volumes, resuspending the oligo dT cellulose is
better, place in 50 mL screw capped polypropylene tube, mix with several
volumes 0.1 N NaOH). REMEMBER, NEVER STORE ANY NaOH solutions in glass ...
always store in plastic, otherwise silicate precipitates will form,
effectively raising the hydroxide conc.!

	The NaOH will cause the oligo dT cellulose to turn yellow ...
don't be alarmed! After several washes in DEPC treated distilled (or well
deionized) water, it will turn white again.

	We routinely reused our oligo dT this way for a couple years,
until we notic that the binding capacity has decreased to inconvenient
levels (determined by how much poly A RNA can be rebound from the first
flow through).

	By the way, to elute mRNA from such columns, try using 55 - 65oC
formamide instead of aqueous buffers. The formamide and heat (we've
compared to water-SDS-buffer, low salt-SDS-buffer, and found the elution
to be quicker (SMALLER volume, sharper peak) and more importantly, the
mRNA is CONSISTENTLY of better quality than when eluted with aqueous
solutions. Formamide, similar to water is polar, thus mixes well with
alcohols, dissolves RNA VERY well (DNA dissolves less readilly), does not
precipitate, protects RNA from nucleases ... it's great.

If using such mRNA for running on gels, dissolve the RNA pellet (obtained
after alcohol precipitation, centrifugation, etc) in formamide instead of
water or other aqueous solution. (Try yourself and you'll surely find
formamide to be the solvent of choice for RNA solutions, except for
enzymatic reactions when the RNA then must be in aqueous solution, then
simply alcohol precipitate, etc, to remove the formamide, then dissolve in
the aqueous solution nedded).

Note added in proof: Regarding RNA and formamide, Chomczynski (who published
several leading articles on RNA extraction techniques) recently published
an article in Nucleic Acids Research (I don't have it with me, but I do
remember that it was a 2 page 1992 article in the methods section). To us
a few years ago, this seemed such common sense that we never considered
publishing on this point.

	Remember to ONLY use formamide that has been "freshly" deionized
(stirred for at least 4 hours with mixed bed resin such as AG 501 X8 resin
from Biorad), can be stored for at least a year in -70oC freezer in aliquots.

Cheers

Andre Hamel
Manitoba Veterinary Virology
545 University Crescent
Winnipeg, MAnitoba
CANADA
R3T 5S6

tel: 204-945-7630
FAX: 204-945-8062
email: hamel at ccu.umanitoba.ca



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