Biomod/2011/TUM/TNT/LabbookA/Concentration measurement of labeled oligonucleotides
From OpenWetWare
Contents |
Concentration Measurement Of Labeled Oligonucleotides
We need to determine the concentrations of oligonucleotides for the composition of the folding batches. The first step ist to measure the absorption coefficient of the unlabeled oligonucleotide, then with this coefficient and the known parameters of the fluorophores the concentrations of the labeled oligonucleotides are determined.
Measuring Absorption Coefficients Of Unlabeled Oligonucleotides
All measurements are performed at a nanodrop photometer. Absorptions of three known concentrations are measured, the absorption coefficient is obtained by a fit using the Beer-Lambert law. The pathlength is 1mm, the concentrations of oligonucleotides are 10, 25 and 50µM.
| concentration [µM] | oligonucleotide 100µM [µl] | 10x TBE [µl] | 100mM MgCl2 [µl] | ddH2O [µl] |
|---|---|---|---|---|
| 50 | 5 | 0.5 | 1.1 | 3.4 |
| 25 | 2.5 | 0.5 | 1.1 | 5.9 |
| 10 | 1 | 0.5 | 1.1 | 7.4 |
Concentration Measurement Of Labeled Oligonucleotides
To A260 of the unlabeled oligonucleotide, A260 of the fluorophore labeled nucleotide is added to get the final absorption coefficient of the labeled oligonucleotide. Now with the average of three absorption measurements, we determined the concentration of the labeled oligonucleotide.
