Poly(A)+ isolation tricks

Richard Lightfoot rlightfoot at CELL-SCIENCE.ORG
Thu Mar 12 10:59:00 EST 1998


the method we've been using very successfully over the past 10(!)years
can be found in Biotechniques 6(2) 114-116, 1988. it is applicable in
our hands only for material derived from cell cultures. it involves a
proteinaseK digestion step in an SDS containing buffer followed by
binding to oligo dT cellulose in the digestion buffer, followed by some
spin column stuff using those funky little spin cups that fit into a
ufuge tube. it works real good for us as long as the usual paranoia
about RNases is shifted into overdrive.

for tissue-derived RNA we use a commercial reagent like TRIZOL followed
by the liquid-phase binding described in the article above. I have found
it necessary to perform a second round of phenol:CHCl3 extraction when
getting the junk from kidney. be sure you use acidified phenol (pH=about
5.5) if you do that step.

I can supply more details if you wish.

good luck, dick lightfoot



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