Alkali destroys RNA. The very fact that it has the free -OH (2') group makes it susceptible to alkali (the phosphodiester backbone is cleaved easily by alkali). Indeed, one way to remove any RNA contamination is to treat/wipe with alkali (the alkaline lysis protocol is therefore handy in giving very RNA-free DNA preps. What runs at the presumptive "100bp" region on the gel is mostly degraded DNA and a lot of oligonucleotides and nucleotides. I am wondering where you got the idea/protocol for RNA isolation using alkaline lysis?
Hiranya S. Roychowdhury, Ph.D. Associate Professor Health & Public Services NMSU-Dona Ana Community College 575 527 7725 (office) ________________________________________ From: Ed Siefker [[email protected]] Sent: Friday, March 08, 2013 8:55 AM To: Hiranya Roychowdhury Cc: [email protected] Subject: Re: LiCl precipitation? Spec reading tells me I got about 150ug of nucleic acid, and I'm sure that's not all plasmid DNA. Running it on a gel shows a little bit of plasmid DNA, and a large bright splotch running around 100bp. No genomic DNA is stuck in the wells. I figure that blotch either has to be RNA, or DNA. If my lysis was harsh enough that it degraded the DNA to 100bp fragments, I would expect to see the small amount of plasmid as a smear instead of a well defined band. On Thu, Mar 7, 2013 at 9:42 PM, Hiranya Roychowdhury <[email protected]> wrote: > How do you know that you got RNA? Alkaline lysis does not give you globs of > RNA. > > > Hiranya S. Roychowdhury, Ph.D. > Associate Professor > Health & Public Services > NMSU-Dona Ana Community College > 575 527 7725 (office) > > ________________________________________ > From: [email protected] > [[email protected]] on behalf of Ed Siefker > [[email protected]] > Sent: Thursday, March 07, 2013 2:29 PM > To: [email protected] > Subject: LiCl precipitation? > > I did an alkaline lysis miniprep and ended up with gobs and gobs of RNA. > I added an equal volume of 4M LiCl, chilled at -20C for 40 minutes, and > spun at 16K Gs for 20 minutes. I don't see a pellet. What am I doing wrong? > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
