rna prep corrections

news at pnfi.forestry.ca news at pnfi.forestry.ca
Thu Jun 17 15:41:08 EST 1993


In article <9306091813.AA13232 at net.bio.net> 22313MGR at MSU.EDU ("Pamela.Green") writes:
>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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