DNA quantitation : what a pain

Krzysztof Wasowicz wasowicz at moskit.art.olsztyn.pl
Mon Mar 2 04:59:05 EST 1998


weazel at animal.blarg.net (Beth Wapelhorst) wrote:

>
>It can be variable, but it shouldn't be THAT variable. Your DNA might be
>too concentrated - and if you're working with genomic DNA, sometimes it's
>hard to get it all in solution - or your spec is on the fritz and needs
>its deuterium bulb replaced.
>
>beth
>DNA Hacker's Resource
>www.blarg.net/~weazel/DHR/dhr.htm
>
>
>
>jsg at mole.bio.cam.ac.uk (J.S Good) writes:
>
>>Hi
>
>>When quantifying the amount of DNA in solution, I usually use the OD260
>>reading in a spectrophotometer. I sometimes find it to be variable -
>>taking the cuvette out and putting it in again can completely change the
>>reading. Do you have any tips for getting more accurate and reproducible
>>readings? Are ELISA kits based on anti dsDNA antibodies available?
>
>>James
>-- 
>sugar beth wapelhorst              "I don't think I'm alone when I say I'd
>weazel at blarg.net                    like to see more and more planets fall
>www.blarg.net/~weazel               under the ruthless domination of our
>                                    solar system." - Jack Handey
One possible problem which I also encountered may be just not enough
DNA solution in the cuvette. I am using Pharmacia's Ultrospec III and
I bought cuvettes for which, I was told, 1 ml of solution is enough. I
had big problems with measurements until I switched  the machine to
550 nm and checked with a piece of paper the path of light. The light
beam was passing just under the top of the solution in the cuvette.
Depending on the position of the cuvette the beam was "scratching" the
meniscus affecting the readings. Using 2 ml of solution removed the
problem.
I had identical problem with a micro 50 microliter cuvette. 50
microliters was not enough to lift the top of the solution out of the
light path. I had to use 70 microliters.
Maybe the reason for the problem is somewhere else (the deuterium
bulb) but it may be good to check the cuvettes also.


Krzysztof Wasowicz
Agricultural and Technical University
Faculty of Veterinary Medicine
Dept. of Animal Anatomy
Bldg 105J
10-718 Olsztyn-Kortowo, Poland
Phone: 4889-5233688, 4889-5233733
Fax:   4889-5234986
Email: wasowicz at moskit.art.olsztyn.pl



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