RNA REMOVAL: Help!
DRHOADS at MERCURY.UARK.EDU
Fri Aug 26 08:08:25 EST 1994
> To: methods-and-reagents at net.bio.net
> From: ez001427 at dale.ucdavis.edu ()
> Subject: RNA REMOVAL: Help!
> Date: Thu, 25 Aug 1994 23:50:08 GMT
> Dear Netters,
> I could use some help in removing trace quantities of RNA from a
> pBluescript plasmid prep that I have. I made a very large (15 mg) prep
> by traditional means, and used a combination of RNASE A and RNASE T1 to
> chew up the RNA. I thought that that would be sufficient, but it turns
> out that the small amount of tiny (runs below 200 bp vs. dsDNA ladder)
> RNA remaining is a problem. So I used a CsCl2 gradient, which removed
> 90 % the RNA, but still there is enough to cause a problem. I would
> appreciate any suggestions for chemically gentle (non-DNA-damaging)
> methods to remove the trace RNA contamination that remains.
> One suggestion I have had is to run a gel filtration column. If
> any of you have experience with such, and could recommend the best media,
> column conditions, etc.. I would be very grateful.
> Thank you in advance for your help, either as a followup post
> here or as email to the address below.
> Marc Goldstein
> Section of Plant Biology
> U. C. Davis
> magoldstein at ucdavis.edu
One method we use that is cheap and very effective is PEG
precipitation according to Lis. (1980) Methods in Enzymology
Mix your DNA solution with an equal volume of 13% PEG
(MW6-8000) 0.8 M NaCl. Incubate on ice 20-30 min. Centrifuge 20 min
at 10-12000 x g at 4C. Carefully decant. Rinse the loose pellet 2x by
addition of cold 70% EtOH, respin for a few minutes. Dry and
redissolve in TE.
This works for DNAs bigger than around 1-2000 bp. Precipitation is
supposed to be strictly dependent on PEG-NaCl concentration and the
molecular weight of the molecule to precipitate. Your RNAs are small
due to the RNAse treatment and should not precipitate.
Doug Rhoads || Dept. of Biological Sciences
drhoads at mercury.uark.edu || 601 Science Engineering
drhoads at uafsysb.uark.edu || University of Arkansas
501-575-3251 || Fayetteville, AR 72701
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