RNAse showing up in gel after plasmid miniprep

Bernard Murray bernard at elsie.nci.nih.gov
Tue Dec 17 12:26:26 EST 1996


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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