To begin, prepare DNA's one treated AAV sample and the dd_PCR master mix. Add 2.2 microliters of the diluted sample to 19.8 microliters of dd_PCR master mix in each tube of the PCR eight tube strip and mix well. Transfer 20 microliters of the mixture into the wells contained in the middle sample row of a droplet-generating cartridge.
Then transfer 60 microliters of droplet generation oil to the wells contained in the lower oil row of the droplet-generating cartridge. Place a rubber gasket over the droplet-generating cartridge, and then place it in the droplet generator. After the droplets are generated, use an eight-channel pipette to slowly transfer 42.5 microliters of solution from the droplet generation cartridge to a multi-well PCR plate.
Seal the PCR plate with an aluminum foil cover using a heat sealing machine for five seconds at 180 degrees Celsius. For amplification, place the plate in a thermal cycler with a 96-deep well reaction module and close it securely and run the PCR program. Once done, load the plate into a droplet reader, input the required information into the system software, and start the reading.
A clear separation between positive and negative droplets was observed in the 1D amplitude plot, suggesting a successful measurement. The second 1D amplitude plot showed droplet rain with droplets scattered between the positive and negative clouds, indicating a potential issue with measurement accuracy. The output data from an AAV run showed consistent results when one sample was measured in duplicates at two different dilutions.
