rna prep corrections

Pamela.Green 22313MGR at MSU.EDU
Wed Jun 9 10:05:00 EST 1993


Dear Networkers,

A couple of days ago an RNA isolation procedure appeared on the
network from Candace Timpte that was attributed in part to our
laboratory.  Although the method posted was based on our
protocol, it contains several modifications (the procedure our
lab uses for RNA isolation from tobacco is detailed in Newman et
al., 1993, Plant Cell 5:701-714; the changes that we make for
Arabidopsis are discussed below), and there are some mistakes in
the recipes that should be pointed out.

In the posted method there were mistakes both in the recipe for
the GTC buffer (amt. of sarkosyl stock to add to get 0.5% was
wrong), and the formulation for the 5X MOPS (NaOAc molarity was
incorrect).  Please see the above reference for an accurate
formulation of both.

Also in contrast to the statement in the posting, we use DEPC
treated water for ALL RNA manipulations and have separate
chemicals for RNA work.  We use only baked glassware and
disposable plasticware when handling RNA and feel that these
precautions are necessary to prepare high quality RNA on a
routine basis from plant material.  When the chemistry is
compatible, we autoclave our stock solutions (e.g sodium citrate,
sodium acetate, and EDTA) before use for MOPS and GTC
preparation.

For the gels, we use a much lower formaldehyde concentration than
was posted (we run 1% agarose/ 2% formaldehyde gels), a different
loading buffer (recipe: 3.6 ml deionized ultrapure formamide, 0.8
ml 10X MOPS buffer, 1.3 ml 37% formaldehyde pH > 4, 0.9 ml H20,
0.5 ml 80% ultrapure glycerol, 0.4 ml of a saturated solution of
BPB), and heat the samples at 65oC for 10 min before loading.
Gels are rinsed twice in 10X SSC before blotting.

For Arabidopsis, we generally use about half as much tissue and
cut the volumes of the reagents in half as well.  (Some people in
the lab also add a phenol/chloroform extraction before the last
chloroform extraction and/or include a second chloroform
extraction.)  For certain types of starting materials, where the
pellet size after the first lithium chloride precipitation is
small, another option is to skip the second lithium chloride
precipitation, but this generally results in a loss of purity.

Best regards, Pam Green  22313pjg at ibm.cl.msu.edu or
22313RNA at ibm.cl.msu.edu (lab address)



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