conc.of NA - summary

HYNEK WICHTERLE jpalek at opal.tufts.edu
Mon Jun 20 18:58:00 EST 1994


Summary - nucleic acid concentrations
Message-ID: <20JUN199419582756 at opal.tufts.edu>
Organization: Tufts University - Medford, MA
News-Software: VAX/VMS VNEWS 1.41    

thanks for all responses and here is a little summary:

	- comparing of dots fluorescence
	- radioactive labeling of NA
	- reading OD at 320nm or scanning from 210-450nm

After all I've found, that comparing intensity of bands in agarose gel is 
the best way how to quantify my RNA. (I can't remove all the partly degraded
or not completely synthesized RNAs, which would interfere with the right 
size band).


QUICK RNA ELECTROPHORESIS

	I've found that for the fast RNA checking I don't have to run
a formaldehyde gel. 
	I separated my RNA on normal agarose-TAE gel (+EtBr) and I've got 
great resultes.
I mixed RNA with agarose dye, denatured for 5 min in 95`C and cooled on 
ice for 5 min just before loading.
I've got sharper bands, no artefacts (shadow bands...), separation of
RNA marker is the same like on formaldehyde gel, bands are better stained
and everything was done in 1.5 hr (comparing to x hours, and pain with
formaldehyde).
	May be it is working only for some RNAs (with weaker secondary 
structure) ...


                            Hynek Wichterle 
						(jpalek at opal.tufts.edu)


From: MUNGO at UVVM.UVic.CA

Try dot blots. Make up a solution with known quantities of the nucleic
acid you want to measure (use RNA or DNA, DNA = lambda, RNA = runoffs from
invitro transcription reaction or perhaps tRNA {i haven't tried tRNA}).
MAke up a solution of EtBr in water at 1 ug/ml.  Mix 4 uL of standars with
4 ul of EtBr, same with sample, dot on saran wrap, estimate your sample
concentration on UV light box.  This method works very well and is equal to
spec readings using an ultra micro cuvette.  Dtandars are usually 1 ug/ml to
10 ug/ml.  Problem with method is that it tells you n othing about the
quality of your RNA...
Good luck
Any problems just ask
Mungo

From: "Zeiler, Brian M'bio" <BrianZ at Microbio.LIFESCI.UCLA.EDU>

Hynek Wichterle,  this is in response to to your posting on the
methods-and-reagents billboard.  I use in vitro prepared RNA in pairing
reactions where I must know, with great accuracy, the concentrations of my
RNAs.  To quantitate my RNAs, I add [a-32P]CTP in my reaction, gel purify my
bands, read dpm in a scintillation counter, and do some math to figure out an
exact concentration.  For your purposes, you may want to use only a little
[a-32P]CTP (or UTP) or [3-H]CTP(or UTP), which is much less radioactive.
However, you must separate unincorporated counts and aborted transcripts so
that your dpm is accurate.  Either spin your reaction through a G-50 column
or, more painstakingly, gel purify your band.
Good luck,
Brian Zeiler
brianz at microbio.lifesci.ucla.edu

From: Chris Bush <bush at mv.MV.COM>

I would also suggest you read your 0D320 as background.  When I have a
problem with OD260's moving around, I can often trace it to particulates
in the prep - if I do a complete scan on the prep (e.g. from 210 to 450
nm) I will see that the baseline is shifted up, sometimes a great deal in
these instances.  I have found that centrifugation in a microfuge, and
careful decanting will remove this problem - often I will even see a
pellet at the bottom of the tube which is particulate.  I know that EDTA
(if much greater than 1 mM that is in TE) also interferes with the 260
reading - but I don't remember where it absorbs.  I usually resuspend RNA
in DEPC water anyway.  Hope this helps -
                                                Chris Bush
                                                Schleicher & Schuell
--
Chris Bush  bush at mv.mv.com
Voice: (603)352-3810 days, (603)239-8164 eves, FAX: (603)357-3627
SnailMail: Schleicher & Schuell, 10 Optical Ave. Keene, N.H. 03431




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