RE: LiCl precipitation?

[Hiranya Roychowdhury]

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: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of Ed Siefker [ebs1...@gmail.com]
Sent: Thursday, March 07, 2013 2:29 PM
To: met...@magpie.bio.indiana.edu
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?

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[Ed Siefker]

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.

[Hiranya Roychowdhury]

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 [ebs1...@gmail.com]
Sent: Friday, March 08, 2013 8:55 AM
To: Hiranya Roychowdhury
Cc: met...@magpie.bio.indiana.edu
Subject: Re: LiCl precipitation?

[Michael L. Sullivan]

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.

[Michael L. Sullivan]

DNase-free RNase, that is!

[Hiranya Roychowdhury]

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: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of Michael L. Sullivan [mlsu...@wisc.edu]
Sent: Sunday, March 10, 2013 1:12 PM
To: Ed Siefker

Cc: met...@magpie.bio.indiana.edu
Subject: Re: LiCl precipitation?

[Michael Sullivan]

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

[Siefker, Ed B.]

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: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of Michael Sullivan [mlsu...@wisc.edu]
Sent: Sunday, March 10, 2013 10:47 PM
To: Hiranya Roychowdhury

[Hiranya Roychowdhury]

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: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of Siefker, Ed B. [Edward...@creighton.edu]
Sent: Monday, March 11, 2013 1:55 PM
To: met...@magpie.bio.indiana.edu
Subject: RE: LiCl precipitation?

[Hiranya Roychowdhury]

I agree. That was a myth that might have been created by careless grad students :)

Hiranya S. Roychowdhury, Ph.D.
Associate Professor
Health & Public Services
NMSU-Dona Ana Community College
575 527 7725 (office)

________________________________________

From: methods...@oat.bio.indiana.edu [methods...@oat.bio.indiana.edu] on behalf of DK [d...@no.email.thankstospam.net]
Sent: Thursday, March 14, 2013 11:02 PM

To: met...@magpie.bio.indiana.edu
Subject: RE: LiCl precipitation?

In article <mailman.164.136305...@net.bio.net>, "Siefker, Ed B." <Edward...@creighton.edu> wrote:
>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.

Just FYI: that's a myth. I used to routinely purify plasmids
> 150 kbp and they elute fine from standard silica columns
with a hot (60-70C) 1 mM Tris pH 8.5.

DK