RNAse showing up in gel after plasmid miniprep

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Dec 30 14:44:00 EST 1996


At 16:17 12/19/96 -0800, you wrote:
>At 04:50 PM 12/18/96 GMT, Paul N Hengen wrote:
>>Marianne Leverone ", BIO (leverone at CHUMA.CAS.USF.EDU) wrote:
>>
>>> Berrnard M:  We are using <1 ug/ml.  Only one person in our lab is having


>>Add MORE RNase. What you are seeing on the gel is the RNA and not RNase!
>>--
>>**************************************************************************
*****
>>* Paul N. Hengen, Ph.D.
/--------------------------/*
>
>But RNA runs well below the 0.5kb marker band, doesn't it? If I recall
right, the band Marianne >originally wrote about is at 0.5kb. Although, I
have to admit, I have never seen RNase band in any of >my gels (and I have
run thousands by now)!!
>
>Dr. Hiranya Sankar Roychowdhury

Hi:

Yes, but...  using RNAse at (per the quote) "<1ug/ml" is a somewhat low
concentration.  It would depend upon *how long* the incubation was at that
concentration of RNAse.   A quick scan of the various non-kit mini-prep
protocols that I have, use as little as 20ug/ml and upwards of 150 ug/ml of
RNAse depending on *where* in the protocol the it is added and then how
long it is incubated.  At less than a 1ug/ml, I'd likely not touch the DNA
for at least 1-2 hours.  I have one protocol where the RNAse is added as
the very last step, so while I'm conducting a digestion, so is the RNAse.

I do find the .5 kb band (smudge) of concern.  At first thought with less
than 1ug/ml of RNAse, an incomplete digestion of RNA comes to mind.
However, if one assumes it is RNAse, how would one be observing it?  Does
EtBr bind proteins as well as it does DNA?  I could be wrong but it would
be news to me.  Nowhere in Maniatis, or Methods of Enz (V152) does it say
that EtBr stains proteins.  (IF it did, we likely could not see our DNA for
all the protein staining in our mini-preps.  Besides, *one* phenol/clfm
will likely not remove *all* protein...)  

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...

In any case, I tend to think the problem is too little RNAse, either in
amount and/or incubation time.  In looking over my mini-s, the RNA usually
runs just in front of the 72 bp Phi-X-HaeIII marker on a 0.8-1% gel.

Just my $0.02 worth.
David

=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
" Sometimes you're the windsheild, sometimes you're the bug."
=============================




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