Good to know that your problem is solved. I surely can relate to these nagging questions. However, as I pointed out earlier, NaOH completely destroys RNA. So, you would not have sen any pellet of RNA anyway. RNA contamination and degraded DNA would have messed up your absorbance data.
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 Siefker, Ed B. [[email protected]] Sent: Monday, March 11, 2013 1:55 PM To: [email protected] Subject: RE: LiCl precipitation? Sorry about the confusion! I was trying to purify plasmid DNA. These plasmids are 15K long and don't elute well from typical miniprep columns. So I have to do it the old fashioned way. I did eventually get rid of the RNA with RNAse, which I actually ended up doing twice to get rid of it all. Precipitating some of the RNA would have helped with that, but I guess they were too short. It still seems strange to me that nothing would have precipitated. If even 5% of the RNA precipitated, I would have seen a pellet. But I solved my problem so I'm happy, thank. -Ed ________________________________________ From: [email protected] [[email protected]] on behalf of Michael Sullivan [[email protected]] Sent: Sunday, March 10, 2013 10:47 PM To: Hiranya Roychowdhury Cc: [email protected] Subject: Re: LiCl precipitation? Well Ed can clarify. His original post is somewhat ambiguous: he didn't really say what he wanted, just that he wanted to precipitate the RNA. It sounded to me like he wanted to get rid of RNA from a plasmid miniprep, especially with his follow up post. But I agree that if he wanted high quality RNA, alkaline lysis is not an appropriate method. Sent from my iPhone On Mar 10, 2013, at 10:10 PM, Hiranya Roychowdhury <[email protected]> wrote: > Looks like you misunderstood the original post. He wants to get RNA from an > alkaline prep. That is not possible. > > > 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 Michael L. Sullivan > [[email protected]] > Sent: Sunday, March 10, 2013 1:12 PM > To: Ed Siefker > Cc: [email protected] > Subject: Re: LiCl precipitation? > > DNase-free RNase, that is! > > 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 > > > > _______________________________________________ > 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
