DNA quantification by fluorometry

Oswaldo oswaldo at iats.csic.es
Mon Aug 7 10:33:16 EST 2000


I use fluorometry to quantify DNA routinely. There are many factors
which can be causing the inconsistency of your readings. Some keypoints
which can cause inconsistency are, in my experience:


- Let the lamp warm up by turning on the machine about 30 min. befor
calibration.

- Have enough sodium chloride in the buffer. You MUST use TNE as
recommended in the machine's operating manual. This manual is available
at Amersham Pharmacia Biotech's website.

- Be sure that you are diluting the 2ul samples well in the 2 ml assay
solution.  You need a reliable micropipette used with good pipetting
techniques. When mixing, avoid air bubbles.

- Always use the cuvette with the same orientation, the one that you
used for zeroing and calibrating. Fluorometry cuvettes (glass) normally
have a mark in one side useful for this orientation. Plastic cuvettes
are theoretically OK with Hoechst dye but in my experience they should
not be used when you need accurate readings.

- Through cleaning of the cuvette is not necessary. I only rinse it
twice or thrice with ddH2O between readings and dry it with paper.
However, avoid fingerprints in sides and bottom of the cell.

If none of these factors are involved then you might have a wrongly made
dye solution or a burned lamp.

Hope that this helps. To me, this little machine is wonderful for
accurate DNA quantification. Consistency between readings is one of the
good things about it.

Oswaldo  

  

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
> 
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