RNAse showing up in gel after plasmid miniprep

Jill Kellogg jkellogg at epitope.com
Wed Dec 18 20:14:57 EST 1996


Hi netters,
Here's another perspective on this subject:
We use RNAse in the first step of alkaline lysis method (100 micrograms per mil
in the cell resuspension buffer).  We incubate our plasmid minipreps 5 minutes
in each of the 3 solutions.  This is faster than adding the RNAse at the end of
the procedure and the RNAse supposedly gets left behind with the rest of the
protein/white junk.  We don't see RNAse migrating in the gel, though we do some
times see single stranded transcript from plasmids with T7 promoters.
_______________________________________________________________________________
Subject: Re: RNAse showing up in gel after plasmid miniprep
From:    ,leverone at chuma.cas.usf.edu ("Marianne Leverone ", BIO) at INTERNET
Date:    12/18/96  9:07 AM

Berrnard M:  We are using <1 ug/ml.  Only one person in our lab is having
this trouble which is wierd.  We can't figure out why he is having such
trouble!  He is now trying a phenol:chloroform then chloroform extraction
to see if he can get rid of it.  this would be a pain because these are
minipreps, and the templates (a lot of them) are being used for DNA
sequencing (for mutation screening).  Marianne

On 17 Dec 1996, Bernard Murray wrote:

> In article <Pine.SOL.3.95.961217105000.7159A-100000 at chuma>, 
> leverone at CHUMA.CAS.USF.EDU says...
> >
> >Hi Group!  When we run plasmid minipreps (alkaline lysis), we add RNAse
> >after the white protein/DNA junk has been pelleted and removed from the
> >neutralization step.  After a 30 min incubation, the plasmid is then ppt
> >with isopropanol and 70% etOH washed and dried and resuspended in water.
> >Then a gel is run to check for complete RNA removal.  Some of our labgroup
> >people are having trouble getting rid of the RNase becasue it shows up as
> >a band (around the 0.5 kb marker) and we have now run enough controls
> >and other tests and have determined that the band is RNase.  ANyone else
> >have this problem and how did you solve it?  Thanks.  Marianne
> 
> I suspect that you are using *way* too much RNase.  If this treatment
> is simply to get rid of the smear to aid visualisation on a gel you
> don't have to use very much at all.  If you need RNA free DNA you might
> try LiCl or PEG to differentially precipitate the two of them.  If you
> have to get rid of large quantities of RNase then phenol/chloroform
> is probably the best way - isopropanol precipitation will help remove
> the ribonucleotides produced by the digestion but the RNase itself
> will almost certainly co-precipitate with the plasmid.
>  I don't see any problems with ethidium staining of gels run with
> aliquots digested with 0.1 mg/ml RNase A (final) and you can probably
> go lower than that.  How are you staining your gels? - maybe other
> methods are more sensitive to protein contamination.
>  Good luck,
>   Bernard
> 
> Bernard Murray, Ph.D.
> bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
> 
> 
> 
> 




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