Accurate quantification can be done by determining the absorbance at 260 nm of a diluted aliquot of the oligo. To do this, first dilute an aliquot of the resuspended oligo in water to a final volume of 1000 µl. Calculate your dilution factor from this step. For example, if 50 µl was diluted, the dilution factor would be 1000/50 = 20. Vortex. Read the absorbance of the dilution at 260 nm. To calculate the concentration of the oligo from the absorbance reading, multiply the absorbance by the dilution factor and then multiply again by the weight per OD (roughly 30 µg/ml).
Approx. concentration (µg/ml) = A260 x 30 µg/ml x dilution factor
For a more accurate quantification the Extinction Coefficient can be used:
Concentration (M) = A260 / E260 (L/mole.cm)
where A260 is measured in a 1cm path length cuvette.
GeneWorks uses the following formula to calculate melting temperature (Tm) of oligos:
Tm = 81.5 + (16.6 x log[Na+]) + (0.41 x %GC) - (675/oligo length)
using a default value of [Na+] = 0.1M
NOTE: There are a wide variety of algorithms available for estimation of oligo ‘melting temperature’. Estimates can vary significantly due to differences in formulae and default salt concentration etc. Oligo Tm does not generally equate to annealing temperature in a PCR reaction. We advise customers to stick to one algorithm and empirically evaluate the relationship of Tm to optimal annealing temperature.