Hi Alexandre,

During my days in academia, I synthesized oligos using both Expedite 8909 and the ABI 394. We used basic spectrophotometric readings to determine the concentration of our oligos and this seemed to work fine. With this in mind, I would favor measurements using the Nanodrop. The Nanodrop can provide a scan of the quality of the oligo which should be a Gaussian curve with a peak at 260 nm. However, there are two issues to consider when measuring absorbance with the Nanodrop.

First, what media will the oligos be measured in, water or buffer? If they are in water then the Nanodrop can also be blanked with water. But if they are in buffer, you may need to likewise blank the Nanodrop with the same buffer for more accurate results.

The second issue is the concentration of the oligos. Spectrometric readings whether using the nanodrop or any other spectrometric instrument has a limited range. You may need to dilute the oligos, then make an additional dilution in order to get a more accurate reading. You could make the final dilution in water and avoid the need to blank with buffer. I would recommend that the absorbance values be limited to 0.2 to 0.8 for the best accuracy.

Hi Doug, thanks for your answer. It helped but I still have questions. With the absorbance value, you simply convert to concentration using the Beer-Lambert equation? But then, which extinction coefficient to use? Or am I being too picky and should just go with the plain, old approximation of 0.33?

We generally used the plain old approximation of 0.33. I would think for most applications, this value would be sufficient to calculate concentration. Although many of the oligo manufacturers seem to favor the nearest-neighbor method of calculation particularly for shorter oligos. It seems to be a bit more involved without aid from a pre-made program. I included a link to a pdf that seems to provide some good information on quantifying oligos.

http://www.ecoli.sk/files/documents/quantification.pdf

I hope this helps.

Hi, Alexandre, if you have synthesized your oligo, you can use the nearest-neighbor method for calculating the absorbance coefficient. You can use pages like this one http://www.basic.northwestern.edu/biotools/oligocalc.html

to do the calculations, simply write your sequence. This coefficient change only minimally if you use a fluorescet tag or have modified bases provided that the oligo is sufficiently long. The value of 0.33 is good if you use random DNA and very long sequences.

I tested the nanodrop in my lab. and agree with Dr. Blintzler: the shape of the drop (hence its volume) changes with buffer, dilute your sample with pure water and try to get a liner response.