Amount	Reagent
5 µL/well	2x Master Mix
0.05 µL/well	Reference Dye
500 nM final/well	forward primer
500 nM final/well	reverse primer
between 10 and 100 ng/well, we used 40 ng/well	template cDNA diluted in a Template Dilution Buffer (1/100 dilution of this buffer was made in nuclease-free water and used to dilute the template cDNA). Diluting the template in this Buffer allows the visualization of the wells where template is added as the reaction color changes from blue to green
nuclease-free water to final volume of 10 µL/well	nuclease-free water