Quanti - iT™ Pico Green dsDNA Assay ( Invitrogen P7589 )
Warm Quant - iT PicoGreen reagent to room temp in the dark .
Prepare 1XTE buffer from 20X stock solution using nuclease - free water : will need 200 μl / well ( for diluting standards , samples and PicoGreen ) .
Dilute DNA standard to either “High” 2 μg / mL ( 1 : 50 of λ DNA stock ) or “Low” 50 ng / mL ( 1 : 1000 of λ DNA stock ) .
For the standard curve , make serial dilutions using the appropriate DNA standard .
Determine amount of sample to assay ( eg , 2μl sample in total of 100μl TE buffer ) .
Add correct amount of TE buffer to all wells .
Add standards to wells .
Then add samples to wells .
Dilute PicoGreen 1 : 200 in TE buffer and protect from light until ready to add to plate .
Add equivalent volume ( 100 μl ) of diluted PicoGreen to every well ( keeping plate in the dark as much as possible ) .
Tap plate to mix .
Incubate 5 minutes at room temperature keeping plate in the dark .
Take fluorescent readings using 485nm excitation and 535nm emission filters .
Determine standard curve and calculate concentration of DNA in samples ( see table in the guidelines ) .
