DNA quantification by fluorometry

Justin Hopkins zmga2150 at kcl.ac.uk
Mon Aug 7 05:11:19 EST 2000


Hi,

I have used this instrument before, and found it to be very reliable and
easy to use. Sadly, we don't have one now, but I'll try and see if what
I can remember may help.

The variation between reading is most probably due to your DNA not being
properly dissolved. This is especially a problem if you are working with
quite concentrated DNA samples.  I think the dynaquant is good for
measuring concs up to around 500ng/µl, but I used to always quantify
much more diluted stocks (maybe 1:20 of original prep), so it's
important to make sure these dilutions are made accurately and
homogenized.

Using freshly prepared dye solution, I would calibrate with 2µL DNA
standard, then repeat with the standard to check the calibration. Then
add my test samples sequentially in duplicate, recording the change in
[DNA] until the machine reached it's upper accuracy limit (1000ng/µL??).
Then, replace the assay solution, recalibrate, check, and continue with
samples. 400 samples would be maybe 10-20 hours tedious work! Rather you
than me ; ) but it is very nice to have such reliably quantified DNA as
it will make your downstream experiments very consistent.

Maxy wrote:

> Hi,
>
> I have over 400 DNA samples to quantify very accurately. I am
> using a flurometer, DYNA Quant 2000 from Hoefer along with
> Hoechst dye for this purpose. Is there anyone out there with
> experience on the use of this instrument? My main problem is that
> I get large variation for repeat measurement of the same sample.
> I also find this method to be very slow.  Are there any other
> ways to quantify DNA very accurately?
>
> Thanks
> max (mariaseg at hotmail.com)
>
> Maxy Mariasegaram
> PhD student
> University of Melbourne, Australia
> E-mail: mariaseg at hotmail.com
>
> -----------------------------------------------------------
>
> Got questions?  Get answers over the phone at Keen.com.
> Up to 100 minutes free!
> http://www.keen.com

--
Justin Hopkins
Cancer Genetics Laboratory
8th Floor, Guy's Tower
Guy's Hospital
London SE19RT






More information about the Methods mailing list