RNA REMOVAL: Help!

Dongxian Yue dxy at iastate.edu
Tue Aug 30 23:09:53 EST 1994


Try extract your DNA with acid phenol. Put your DNA in 0.3M NaAC, pH4.3,
o.75M NaCl, 10 mM EDTA, Add equal Vol. of H2O saturated phenol, Vortex.
ALL RNA will go to the H2O phase (top), DNA will be in the phenol
phase. Then add 2M tris base to nutrolize the phenol, Now  your DNA will
be in the upper phase. This is way you do not need to bother with CsCL
and RNases. 

My work also requires large amount of plasmid in order to transcribe
30mg RNA very often, RNA or RNAse in my plasmid prep will be fatal for
me. This is how I get rid both of them After tried all other methods.



Dongxian Yue

dxy at iastate.edu

.


In article <Cv47JK.Mwt at ucdavis.edu> ez001427 at dale.ucdavis.edu () writes:
>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
>










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