DNA quantification

[Page Briscoe]

sjwhite at ksu.ksu.edu (Susan Jane White) wrote:
>
> I'm purifying genomic DNA from a filamentous fungus. I want to know how much
> I have, but I suspect I have too little (or of too bad quality) to get good 
> spec readings. I tried using EB and spotting onto Saran wrap on a lightbox
> (ref. Sambrook et al.); this seemed to work OK, but the second time my standard
> curve was pretty much all the same - I'm not sure if they got concentrated by
> evaporation in the fridge or what. Does anyone have any easy, reliable method?
> I've seen ads for a "Dipstick" DNA assay (Invitrogen?) but it was pretty
> expensive and they wouldn't send me a sample (hmpf...). Are they nice? Is there
> an easy "home-made" version?
> 


We have had problems with our spectrophotometer, so we had
to find a way to estimate our DNA concentrations without it for
a while.  What we did was take a sample of isolated DNA and run
it on a gel to get an estimate of the amount and purity of the
isolated DNA.  I have also tried using the DNA Dipstick kit.
It works fairly well if you only process 2 to 4 samples at a 
time.  It uses a cuvette as the reaction vessel, so I do 2 at
a time and face the sticks back to back.  The sensitivity is claimed
to be as low as 0.1 ng/microliter.  The difficulty I had with this
kit was actually esitimating to my satisfaction the amount of DNA
I had.  The color makes it difficult to read in between 0.1 ng/microliter
and 0.5 ng/microliter.  The ranges are: 0.1, 0.5, 1, 5, and greater
than 10 ng/microliter.  Also, this kit does not give you an idea
of the purity of your DNA.  The cheapest thing to do is to run a minigel
with small wells (for us, that's what we do).

I hope this helps.

Page Briscoe
The Gene Therapy Program
University of Alabama at Birmingham
Birmingham, Alabama