RNA REMOVAL: Help!

Michael van Waes bi__mvw at SELWAY.UMT.EDU
Fri Aug 26 12:28:27 EST 1994


>
>	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
>
>
>
 How about alkalilne hydrolysis? Just raise the pH to 10 and incubate at 65
 degrees for 30' or so. That's sure to digest the RNA without affecting DNA

 Just a thought. Good Luck!



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