Determine Concentration and Label Efficiency

Take an UV/Vis spectrum using quartz cuvets from 240 to 650 nm. Dilute the samples if necessary to be in the linear range of the spectrometer (usually 0.2-0.5 OD).

3.3.1. Calculating the Labeling Efficiency and Concentration of Oligonulceotides

1. The extinction coefficient of oligonucleotides can be estimated by:

£Oligo + (#G ■ 11,500 + #A ■ 15,400 + #C ■ 7400 + #T ■ 8700) M-1cm-1 where #G, #A, #C, and #T are the numbers of guanosine, adenosine, cytidine, and thymidine, respectively.

2. Correct for contribution of the dye to the absorbance at A260 (see Fig. 2).

AoUgo = A260 - Adma ■ CFdye whh CFdye = £™ J^ye The correction factor CFdye can be determined from the absorption spectrum of free dye. For fluorescein and TMRh this value is approx 0.3.

3. The labeling efficiency is given by:

Dye:oligo = (Aoligo ■ £^0/^7 • W) The extinction coefficient £dye of the bound dye can vary from the value of the free dye. In the case of 6-fluorescein the extinction coefficient decreases by approx 25% from 81,000 to 60,000 M-1cm-1, whereas for 5-TMRh it is nearly unchanged (decreases slightly from 80,000 to 75,000 M^cm1).

wavelength / nm

Fig. 2. Absorption spectrum of a 20-bp long double-stranded DNA completely labeled with 6-fluorescein and 5-TMRh at the helical ends. The spectra of free fluorescein (O) and 5-tetramethylrhodamine (A) show the contribution to the 260 nm peak where DNA absorbs.

wavelength / nm

Fig. 2. Absorption spectrum of a 20-bp long double-stranded DNA completely labeled with 6-fluorescein and 5-TMRh at the helical ends. The spectra of free fluorescein (O) and 5-tetramethylrhodamine (A) show the contribution to the 260 nm peak where DNA absorbs.

4. The concentration can be determined by:

Coligo Aoligo/Eoligo

3.3.2. Calculating the Dye-to-Dye Ratio and Concentration of dsDNA

1. In most cases the absorption spectrum of the donor overlaps with the spectrum of the acceptor dye (see Fig. 2). Although the absorption of the acceptor can be usually measured without any contribution of the donor, the donor signal has to be correct.

^donor ^ ^acceptor ^1 acceptor vv acceptor efree acceptor ^free acceptor

For the FRET pair 6-fluorescein and 5-TMRh this correction factor is Emm/Emm, = 0.095.

2. The dye-to-dye ratio is then given by:

donoraCCeptor (Adonor • Eacceptor)/(Aacceptor • Edonor)

3. The concentration of dsDNA can be calculated as described for oligonucleotides in Subheading 3.3.1. The extinction coefficient of dsDNA is approx 12,000 M1 cm1 per basepair.

0 0

Post a comment