RNA REMOVAL: Help!

Douglas Rhoads 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 
65:347-353.  
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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