Despite a few of the other posts, in my experience, it's not uncommon to have RNA fragments like you describe in a simple alkaline lysis mini prep. Back when I used to do this sort of prep, we always included an RNAse step prior to the final plasmid DNA step to take care of this problem. Omission of the RNase step meant a big blob of nucleic acid when you ran it out on a gel. Similarly, column based plasmid mini preps include RNAse in the lysis buffer. Yes, RNA is not stable at high pH, but my experience tells me it's not sufficient to degrade RNA to fragments not precipitable by ethanol.
Now, as to your question of why you couldn't get rid of the RNA by the standard LiCl precipitation. I've come across several references saying that LiCl is not particularly effective at precipitating RNA that is less than 300 bases. So, I suspect what you have are relatively short fragments of RNA. Why don't you treat with DNAse-free RNA and reprecipitate your plasmids with ethanol? That is, I think, a pretty standard approach to this problem. Good luck. On Mar 8, 2013, at 9:55 AM, Ed Siefker <[email protected]> wrote: > 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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
