Oligo concentration determination - Is this correct? (Sep/17/2009 )

So I received some oligos (50 nucleotides long), but it doesn't say how much per tube (long story). I diluted the oligos in 480 ul of nuclease free water and measured the OD myself by mixing 10ul of each in 990 ul of water and then did some calculations. Here is an example:

Oligo 1: OD260 .5696

Using an online DNA concentration calculator (http://www.pubquizhelp.com/other/dnacalculator.html), I determined the concentration to be 1.71 ug/ul (1710 ug/ml).

I took that and used it with the Promega biomath calculator (http://www.promega.com/biomath/Default.htm) and determined that 1.71 ug/ul of ssDNA equals 103.626 pmol/ul.

Now, if I'm not mistaken 100uM = 100pmol/ul, so I would conclude that the concentration of this oligo is 103.626 uM.

Is this procedure correct? I get confused easily with calculations, so I just wanted to check.

Thanks in advance for any help.

Pancho on Sep 17 2009, 11:21 AM said:

Don't always rely on calculators, you should do these calculations on your own!

The concentration of your oligo based on the absorbance at 260 nm is:
conc (ug/ul) = ((optical density/extinction coefficient for ssDNA)*1000)/ul sample used
The optical density was 0.5696
The extinction coefficient for ssDNA is ~33 (this is less accurate for short oligos or sequences with numerous repeats)
You used 10 ul sample
Thus, conc (ug/ul) = (.5696/33)*1000/10 = 1.726 ug/ul

To obtain the molar information from your concentration, you will need the molecular weight...there is no direct conversion from just "ssDNA." What is the MW of your oligo? or if you don't know, you can determine an average MW from the number of base pairs (660 daltons for the average molecular weight of one nucleotide PAIR, or 330 daltons/nucleotide = 16500 daltons (g/M) for 50 bp).
Based on your initial resuspension of the oligo in 480 ul and that you used 10 ul for the OD reading, you should have 470 ul left at 1.726 ug/ul for a total of 811.22 ug of oligo.


A 1M solution would have 16500g/L or 16.5 g/ml.
Your solution has 811.22 ug in 0.470 ml = 1.726*10^-3 g/ml
Since a 1M solution has 16.5 g/ml and yours has 1.726*10^-3 g/ml, you have:
1.726*10^-3 / 16.5 = 1.046*10^-4 M
or 104.6 uM.

To sum up, based on a formula weight of 16500, with 811.22 total ug, in a volume of 470 ul, your solution has a molar concentration of 104.6 uM.

Good calculator: http://www.basic.northwestern.edu/biotools/oligocalc.html

It's more accurate with extinction coefficient.

Your oligo (50b.p. equal ATGC) is ~111 microM.

+/- 50% in PCR works fine as well