rodriguez at kchf.ch.pwr.wroc.pl
Thu Oct 29 05:46:55 EST 1998
> Hi all (with greetings to Mechthild),
> is it true that quantification of DNA using OD 260 values after Qiaex II gel
> extraction do generally over-estimate the yield? Using gel quantification I
> ended up in about half of the concentration...
> Beat Reidy
> beat.reidy at ipw.agrl.ethz.ch
As someone pointed out, the buffer you use for measurments is critical. In
I always make a double reading A260 & A280. If you used water, the A260/A280
ratio will be sth like 1.5-1.6. When using buffer with a pH over 7 I get an
A260/A280 ratio of 1.7-2.0. I would not reccommend dissolving DNA in TE if you
plan to perform a cycle sequencing on it.
I also noticed that A260 measurements tend to overestimate the yield. I use the
Geneclean III kit from BIO101.
Still, I am not quite sure that your problem lies in buffer choice. When
isolating bands from a gel, I use a Petri dish assayfor concentration
measurements. This is simple: pour 0.8% agarose with EtBr into petri dishes
(they may be stored for up to a month in a cold room) and prepare DNA
concentration standards (10 ng/ul, 25ng/ul, 50ng/ul, 75ng/ul, 100ng/ul, 150ng/ul
and 200ng/ul). Now, to asses the concentration of a sample, you simply take a
dish, make 0.5ul dots of the standards, and similar dots below for your samples.
Wait till the dots dry, and then compare your sample against the standard. This
not a very accurate method, but works. It is very useful when you want to
measure the concentration of a mix of DNAs of different sizes.
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