Quantification of DNA - Genequant?
HW
Genequant device from Amersham? We are not very happy with our old
UV/vis spectrometer and do not confide much in the data it produces.
Is there more equipment on the market that performs well? I know it's
a tough job to reliably quantify DNA but we really need to know the
approximate concentration of our DNA samples.
Any information is greatly appreciated.
Hans
Henk Veldman
If you have access to a fluorometer, you can simply use a dye like
Hoechst 33258.
See for instance:
Labarca & Paigen, 1980
A simple, rapid, and sensitive DNA assay procedure.
Anal Biochem 102:344-352.
Henk
Wolfgang Schechinger
I still prefer "ordinary" full spectrum photometers against the Genequant.
I used a Genequant for some time in parallel to a Gensys photometer and
found the results from the Gensys photometer to be more consistent and
reliable.
I also didn't like the time regime of the Genquant that tells you when to
insert the sample and when to remove it.
The Genequant II that I used was still labelled Pharmacia (sold before they
merged / were eaten by Amersham), so maybe they have improved the device in
the meanwhile.
Compared to the difference in price, if you're not absolutely broke, I
think a tunable photometer is worth spending the money since it offers you
many more possibilities like protein quantification with lowry /BCA style
reagents, determination of concentrations of other things than xNA etc etc
etc. There are lots of brands and models on the market that are fast,
perform very well despite they are low cost and even have a programmable
interface that gives you all the ratios you desire. All you need to
consider is that they can hold microcuvettes with a volume less than 100ul.
All the best,
Wo
---
Robert Whittier
>Genequant device from Amersham? We are not very happy with our old UV/vis
>spectrometer and do not confide much in the data it produces.
>
>
>Is there more equipment on the market that performs well? I know it's a
>tough job to reliably quantify DNA but we really need to know the
>approximate concentration of our DNA samples.
>
>
>Any information is greatly appreciated.
>
>
>Hans
If your only purpose is to measure DNA concentration, I would recommend
using Hoechst 33258 fluorochrome fluorescence. It is very reliable and
sensitive, although it can be affected by the GC/AT ratio of your DNA
(the dye binds poorly to very GC-rich DNA). An advantage is that RNA
contamination gives very little signal, so what you measure is dsDNA. You
can use a full-featured fluorometer, but Amersham also sells a one-trick
pony,
a dedicated fixed wavelength fluorometer that they call the DyNA-Quant 200.
This was originally a Hoefer instrument before various acqusitions and
mergers.
With mammalian or E. coli DNA, 20 ng is plenty for a reliable measurement.
For more info on the method, check Current Protocols in Molecular Biology
(Wiley) page A.3D.3. The original reference is:
Labarca C and Paigen K (1980) A simple, rapid and sensitive DNA assay
procedure. Anal. Biochem. 102: 344-352
I've used this method since before I joined Amersham, and will continue
to use it following my imminent departure from the same.
Bob
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---
Deanne Bell
My experience with DNA sticks, the spec and fluoremeters have proven inconsistent and unreliable. My current method of choice to quantify DNA is to run 3ul of my prep on a 8% agarose gel along with some Molecular MASS Standards (i get mine from BioRad, no affil, other companies may sell them). Stain with ethidium bromide and quantify with spot densitometry.
Saves alot of my prep and I believe it is most accurate.
Hope this helps
DBell
-----Original Message-----
From: HW [mailto:jcw...@removethis.yahoo.com]
Does anyone in this group have any experiences, good or bad, with the
Genequant device from Amersham? We are not very happy with our old
UV/vis spectrometer and do not confide much in the data it produces.
Is there more equipment on the market that performs well? I know it's
a tough job to reliably quantify DNA but we really need to know the
approximate concentration of our DNA samples.
Any information is greatly appreciated.
Hans
---
Wolfgang Schechinger
unfortunately, most PIs insist in doing so, it's probably part of GLP. If
you don't do it and the reaction does not work, then they blame your
laziness and when you do it and it does not work, then they blame your
incompetence.
Wo
At 03:01 23.05.2003 GMT, D.K. wrote:
>In article
<01FC4B0027304840892...@SERVER01.fresno.ars.usda.gov>,
db...@fresno.ars.usda.gov ("Deanne Bell") wrote:
>>I think this is one of those areas where everyone has their own =
>>preference.
>>My experience with DNA sticks, the spec and fluoremeters have proven =
>>inconsistent and unreliable. My current method of choice to quantify =
>>DNA is to run 3ul of my prep on a 8% agarose gel along with some =
>>Molecular MASS Standards (i get mine from BioRad, no affil, other =
>>companies may sell them). Stain with ethidium bromide and quantify with =
>>spot densitometry.
>>Saves alot of my prep and I believe it is most accurate.
>>Hope this helps
>
>Yeah, for sure. That aside, I've seen countless examples of people
>religiously measuring their DNA concentration (plasmids and
>fragments mostly) in cases where it is just about useless.
>
>For 99% of your routine molecular biology, "microliter" is perfectly
>good measure of DNA concentration :-) All it takes is to have an idea
>of plasmid copy number and do ranges of dilutions whatever you are
>doing (applies to PCR, ligation, restriction - the only way to find
>optimum is to try different amounts; books and protocols have no real
>value).
>
>DK
>
>plas
>
>
---
HW
the information as most usefull although I've not decided yet how on
to proceed.
Hans
Jonathan Redfern
just a thought but have you tried the new NANODROP Spectrophotometer (http://www.nyxorbiotech.com). The advantage of this unit is that it uses no cuvette and goes down to 1ul volumes !! the cost is around EUR 9000
good luck
Jonathan
HW wrote
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Joyce Faler
several years old, no printer option, and very slow. I've
also used the next step up Genequant device which has a
printer and can use the "disposable" cuvets - definitely
faster but still very labor intensive. Not familiar with
later versions.
I'm investigating the BioMate 3 (from Thermospectronic,
available from Fisher Scientific) which can be purchased
with a printer option and a sipper option that will
supposedly read 300 samples/hour. If anyone has actually
used this machine, I would really like to hear what they
think of it.
-Joyce Faler