Expected accuracy of UV spectrophotomers

[Josh Perfetto]

Hi,

I'm wondering what the expected accuracy of UV spectrophotometers are when quantifying dsDNA. I had DNA in TE which I believed was 100 mg/ml based on resuspending a known quantity of DNA as measured by a synthesis company. I dilluted it 50x to a total volume of 100ul and measured in two different spectrophotometers.

Spec 1 (Thermo Electron Helios Gamma):

A260 = 0.031 A

A280 = 0.019 A

Calculated concentration: 77.5 ug/ml

Spec 2 (Eppendorf BioPhotometer):

A260 = 0.050 A

A280 = 0.022 A

Calculated concentration: 125 ug/ml

The consistency was a little disappointing. Is this normal or do you think there is a problem with my specs? I suppose I could get more accuracy by diluting less but I hate using up so much plasmid just for quantification.

-Josh

[Simon Quellen Field]

The first sample was 22.5% low.

The second was 25% high.

Eppendorf claims a 1% accuracy.

You can get calibration filters:

"http://www.eppendorf.com/int/index.php?pb=98fcce74085c8923&action=products&contentid=1&catalognode=80177"

Sources of error can be the cleanliness of the cuvette. Fingerprints?

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[shamrock]

I take DNA concentrations as determined via spectrophotometer with a bit of skepticism and verify by electrophoresis.

 

If I had some known standards that would validate the calibration of the spec I might have more confidence in the readings, but I've often seen discrepencies between the spec and electrophoresis results.

[shamrock]

I ran across this in Molecular Cloning 3rd edition volume 3 page A8.20

 

"Note, however, that the extinction coefficients of both DNA and RNA are affected by ionic strength and the pH of the solution (references). Accurate measurements of concentration can only be made when the pH is carefully controlled and the ionic strength of the solution is low."

 

May help explain the variation that I have seen.

[Josh Perfetto]

I was using new disposable UV cuvettes so they were pretty clean. Calibration may certainly help - though it seems standard sets like that are about $2000 which is several times more than I paid for the machine :) I saw some other standards more like $600, I will keep looking.

-Josh

[Sebastian S. Cocioba]

If you want reliable quantification why not try the qubit fluorometer from life? Its a spec made for dna rna and protein and is 1000x more accurate than a nanodrop. It uses a fluorotag reagent that is thermosensitive but incredibly accurate. The machine is 1200 new but its well worth it if quant is of paramount importance. I use it regularly and its awesome!

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Nathan McCorkle]

You might just try calculating the Henderson hasselbach calculation using activity coefficients, which takes into account the ionic strength of the solution.

Doing this with pH calculations makes a decent amount of difference.

I was writing some python code to do this calculation, lemme go finish it up.

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