Nice.
Didymium glass is easy to come by for those who don't need an NIST
traceable calibration source.
But at 5 nm resolution, it is not as good as the peaks from a fluorescent light
bulb (I get sub-nanometer resolution in my $20 spectrograph). On the other hand,
it has lots of nice peaks all through the visible spectrum.
For DNA quantification, we would be looking for something with a peak
absorbance at 260 nanometers. To assess DNA contamination of protein
samples, we would want something with peak absorbance at 280 nanometers.
It turns out that sub-nanometer accuracy is useful for the 280 nanometer
reading (much less so for the 260). The values for the five interesting nucleic
acids given on that page might also be useful calibration targets. If a solution
of guanine shows a 1.15 ratio, and a solution of adenine shows a 4.50 ratio,
then we can be pretty happy with whatever results we get with our sample, for
the 260/280 ratio test.
According to that web page, two different equally accurate (to 1 nm) spectrometers
may disagree by as much as 0.4 on the 260/280 ratio reading of the same sample.
So presumably, the accuracy of a 260/280 test is no better than that, and so half
a significant digit in the ratio is adequate for publication:
Accuracy of test at 1 nanometer resolution:
low mid high
Guanine: 0.75 1.15 1.55
Adenine: 4.10 4.50 4.90
Cytosine: 1.11 1.51 1.91
Uracil: 4.60 4.00 4.40
Thymine: 1.07 1.47 1.87
For those building a DIY spectrophotometer for this test alone, the focus problems
go away and you don't have to tilt the sensor, or even use a 2D sensor. The peak
at 260 is a plateau anyway, so if it is out of focus, we don't care. We need sub-nanometer
resolution only at 280 nanometers, so we focus on that spectral line, and everything
is copacetic. Such a scope can be built in about 15 minutes for around $20, using a
digital camera for the sensor.
And it should be easy to make calibration cuvettes from pure nucleic acids.
You don't care much about how concentrated the sample is -- you just care
about the ratio of absorption at 260 and 280 nm. But pure samples of nucleic acids
are easy to come by, and can be used for the non-ratiometric calibrations we were
discussing earlier.
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Get a free science project every week! "http://scitoys.com/newsletter.html"On Sun, Nov 27, 2011 at 10:07 AM, Tom Knight <tk@csail.mit.edu> wrote:
You want a didymium perchlorate solution as your wavelength standard. Check out the sharp peaks in its spectrum:
http://www.starnacells.com/d_ref/d_wl/DL.html
You could check out some of their other reference standards on the left.
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