Sandie,
I can suggest two methods for high volume DNA quantification.
A. OD260 in multiwell plate readers.
Multiwell (especially 96-well) UV/VIS plate readers are sold by many
companies/distributors, in addition you would need some multichannel
pipettors, or better a robot, to prepare the DNA dilutions in those plates.
An ELISA washer would help, too.
Ask the customer/DNA supplier to prepare you the dulutions in multiwell
plates, or at least to have aliquots of his/her undiluted DNA samples in
96-well format, so that your technicians do not lose time pipetting well by
well.
B. Spotting on an agarose gel with EtBr.
Do not have the funds for the machines? The 96-well plates can help,
anyway. A low-tech solution is to prepare standards of DNA (from 10 to 200
ng/ul, in 25 ng increments), spot 0.5 ul of those samples on an agarose gel
with EtBr, then spot 0.5 ul of the DNA smaples from your 96-well plates on
the same gel, and wait 1 to 5 minutes. The surfice of the gel must be dry,
not wet, so that the samples do not spread on the surface but get absorbed
fast into the gel. Take a picture from a UV transilluminator, and determine
the concentrations of the samples by eye comparison with the standards
(there are smarter ways with picture scanning, but that is another story).
This method is less exact than the OD260 (e.g. you may determine that the
concentration is between 75 and 100 ng/ul), but you may not need exact
quantification for your sequencing templates. On the other hand, the
spotting has three major advantages: 1) Cost. You do not need to spend $20K
(or much more) on a UV plate reader and later on UV-transparent plates,
lamps etc. 2) Sensitivity. The UV reader needs hundreds of times higher
volumes than the 0.5 ul needed for the spotting, as a rule you are forced to
measure DNA dilutions on the UV reader, therefore for some DNA samples wil
give you OD readings well below 0.100, which cannot be used with confidence.
The spot method may use undiluted DNA and the sensitivity (5-10 ng/ul) is
much better than the UV reader. 3) Discrimination against nucleotides and
short RNA/DNA junk. Most of the DNA extraction methods rely on RNase
digestion to remove the RNA contaminants from the DNA sample. The RNA may
not always be completely digested (partially double-stranded RNA structures
are more resistant, remember the tRNA structure?), in addition you may have
completely degraded DNA, i.e. low m.w. DNA junk, and even the completely
digested RNA (the nucleotides) will give you high readings on OD260 and will
lead to false determination of "high" amounts of DNA by the OD260 method.
The spot method will not take into acount small pieces of DNA/RNA or single
nucleotides, because those will not absorb (or will absorb very little)
EtBr.
I am talking here about spotting as a method for quantification of genomic
DNA, phage/BAC/cosmid DNA, plasmid DNA, or PCR products, i.e. higher m.w.
(at least 50-100 bp) DNA, not oligos (many oligos will not abosrb EtBr at
all).
Regards,
victor
www.alphadna.com
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Sandie Smith
Sent: July 26, 2000 16:21
To: Recipients of ABRF List
Subject: DNA Quantitaion
I work in a DNA Core facility and would like to quantitate multiple DNA
samples for sequencing using AB's big dye. We currently have two 377s and
will be getting a 3700 in the middle of August. I would like suggestion
for quantitating many DNA samples.
Thanks.
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