Fluorometer: to buy or not?

Christopher T. Cole colect at caa.mrs.umn.edu
Mon Feb 12 17:24:25 EST 1996


On 9 Feb 1996 15:13:37 GMT, 
Stephen Marcus Lane  <smlane at unity.ncsu.edu> wrote:

>I'm considering whether or not to get a fluorometer for the
>quantification of DNA (and RNA, if possible).  I've only
>looked at the Hoefer DyNA Quant 200 so far, and it looks
>like the price will be around $3000 for the meter and 
>supplies.  
>
>I really want to have a more accurate and sensitive way
>of quantifying DNA than spectrophotometry in a 1 mL cuvette.
>I just don't trust it.  The DNA samples that I want to quantify
>run the gamut; PCR products, cell preps, other polymerase/ligase
>/nuclease reactions, all in various forms of purification,
>by EtOH or phenol/CHCl3 or agarose/acrylamide gel elution
>(yes, I'm trying out a lot of different techniques).
>
>I know mononucleotides, proteins, and phenol pretty much render
>an A260 measurement worthless if they're present in even small
>amounts.  And I think the same is true for agarose and
>acrylamide (?).  So I don't like relying on spectrophotometry.
>
>I haven't tried using a 4-variable equation at 230, 260, 280,
>and 320 nm.  Can anyone offer info on the improvement this
>offers over just A260 (and A260/A280) for possibly-contaminated
>samples?
>
>One final thing; I've just started checking out fluorometers.
>I can't seem to find anything in the catalogs about what
>contaminants can throw off the reading.  Is the Hoechst assay
>specific enough that the above contaminants (monont's, proteins,
>gel debris) aren't detected?
>
>Thanks for any help, Steve
>--
>-- Steve Lane <steve at ncsu.edu> ------------------------------- 
>
>
Steve:

We got a Dyna Quant 200 last summer and have been very happy with it.  It 
is useful for much smaller quantities and/or concentrations than we can 
handle in 1 or 2 ml cuvettes with UV.  We do not use the capillary 
adapters.  Most of our readings are in the 5 to 200 ng/ml range.  I put the 
device in the hands of undergrads and leave them alone with it, reliably.  

--Chris




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