Quantifying minute amounts of DNA?
nash at nrcbsa.bio.nrc.ca
Tue Feb 22 19:09:36 EST 1994
In article <2kd3t1$rp3 at eldborg.rhi.hi.is>,
Helgi Briem Magnusson <hbriem at rhi.hi.is> wrote:
> Does anyone have a protocol for measuring the amount of
>DNA when you only have a tiny amount available, i.e. too
>little to visualize on an EtBr-agarose gel and too little
>to give a reading at 280nm when diluted?
> Any help would be much appreciated.
>Regards, Helgi Briem
>Inst. Exp. Pathol.
This is the way I quantitate all my PCR reactions, DIG-labelled DNAs
and low-yield minipreps. It is a kit-free (Hi Jim, ED) and cheap
method, and comes from the old Cold Spring Harbor Manual (the
pre-Maniatis one). I could look up the reference for you.
A. Make a litre of 1% agarose in TE buffer. Add 50 ul of a 10 mg/ml
solution of ethidium bromide (Final conc: 0.5 ug/ml). Pour 20 ml
plates. When they have set, wrap them with Parafilm to stop them
drying out and store them in the dark at 4 deg. My last batch lasted
1 year in a cardboard box in the fridge.
B. Make up 1 ml solutions of standard DNA (I use bought lambda DNA)
at 10, 8, 6, 4, 2, 1 and 0.5 ug/ml.
C. Spot 5 ul of the standards on the plates. Also spot 5 ul of
DNA-to-be-measured. I usually also spot 5 ul of 1:5, 1:10 (and if
necessary 1:50 or even 1:100) dilutions of sample. Note: if there is
> 0.01% SDS in the sample, it won't spot, but makes a mess (no surface
tension). I find the best way is to spot the standards in a large
circle about 1.5 cm from the edge of the plate, and the samples in the
D. Gently air-dry the plates (I put mine on the lid of my 60 deg
waterbath for 5 - 10 min). Don't knock the plate!
E. Photograph the plate (inverted <-- gel side down) as you would an
electrophoresis gel - I use Polaroid type 52 film in a Polaroid MP-4
camera set up with a Wratten 22A filter, with a 5 sec exposure.
F. Using the standard curve as a guide, eyeball the appropriate
dilution and estimate the DNA conc of the sample.
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