RNAse showing up in gel after plasmid miniprep

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Tue Dec 31 05:44:32 EST 1996


In article <3.0.32.19961230134614.006890a0 at imm2.imm.uth.tmc.edu>,
dhavilan at IMM2.IMM.UTH.TMC.EDU ("David L. Haviland, Ph.D.") wrote:

> However, if one assumes it is RNAse, how would one be observing it?  Does
> EtBr bind proteins as well as it does DNA?

You are right.  An intercalating agent like EtdBr binding with high
affinity to a protein.  Hardly!  Also, at the concentration 1 ug/ml
assuming that our friend the original poster loads 10 ul on the agarose
gel, we have some 10 ng of RNase.  If EtdBr were that effective at
staining proteins I doubt that we would all be using Coomassie.

> There is a marked difference in the molecular weight of RNAse and 500 bp of
> some_unidentified_nucleic_acid.    The MW of RNAse is 15,000 (Meth. Enz.
> 152:21) and guestimating (Promega manual) each nucleotide (one phosphate)
> at about 324 daltons puts a 500 base fragment at 162,000 - double that if
> double stranded.   Granted I'm mixing nucleic acids and protein so I'm not
> entirely sure the comparison can be made...

This comparison is of course not that simple.  Since a protein would run
according to its pI (or rather net charge at pH 8)  under the conditions,
and not according to molecular weight. But it would anyway be highly
unlikely for RNase to focus as a band in a 1% agarose gel.

> In any case, I tend to think the problem is too little RNAse, either in
> amount and/or incubation time.

I think that would be the logical conlusion.

Happy new year !!

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________



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