Preparation of Viral Sample and Master Mix for Digital Droplet PCR to Accurately Measure the Viral Genome Copy Number

To begin vortex the Adeno-associated virus or AAV sample and centrifuge to ensure all liquid remains at the bottom of the tube. Add 45 microliters of DNase I containing solution to a 0.2 milliliter PCR eight tube strip tube. Transfer five microliters of the AAV sample into the tube containing the DNase solution.

After vortexing, centrifuge the sample briefly to ensure all liquid remains at the bottom. Using a thermocycler, incubate the samples for one hour at 37 degrees Celsius, then cool them down to four degrees Celsius. Next, prepare appropriate dilution of the DNase's treated sample in AAV dilution buffer in a fresh 0.2 milliliter PCR eight tube strip.

After serial dilution, vortex the samples and centrifuge to ensure all the liquid remains at the bottom. Combine the forward and reverse primers probe ddPCR Supermix and DNase's-Free Water in the required volumes. Vortex the master mix and briefly centrifuge the tube.

Then transfer 19.8 microliters of the master mix into each tube on a fresh 0.2 milliliter PCR eight tube strip.