Real time PCR experiences

Real-time reaction mixes

We have tested various reaction mixtures for sensitivity and PCR efficiency by amplifying a 169 bp PCR fragment cloned into a vector which was linearised prior to amplification. The running conditions were as recommended by the manufacturers. 40 cycles were performed and the result is shown below. The various curves represent successive 10-fold dilutions of the standard.

The reaction mixture from Takara is almost comparable to the Roche FastStart DNA Master SYBR Green 1 mix. The only difference we observed was that signals in very dilute sample of the cDNA (10E-9) were undetectable due to dimer formation.

Primer concentration

If your amplification efficiency is not satisfactory, you might want to try to vary the concentration of the primers. We usually use 200 nM final concentration, but both higher and lower concentrations can be tried. Below is an example where the efficiency is increased from 1,65 to 1,82 by increasing the primer concentration from 200 nM to 500 nM.