Q 100 droplet reader

ddPCR was carried out in order to precisely re-quantify the vector genome (vg) titres of the purchased AAV vectors in a side-by-side measurement. The reaction mixtures were assembled with 10 µl ddPCR Supermix (Bio-Rad, Hercules, CA, USA), TaqMan primers and probes (Applied Biosystems, Foster, CA, USA) (final concentrations of 10 µM), and template (5 µl) in a final volume of 20 µl. In order to ensure consistent results, reaction mixtures were prepared with 2 different primers/probes: CMV assay (Forward: 5′-GCACCAAAATCAACGGGACT-3′; Reverse: 5′-CTCCCACCGTACACGCCTAC-3′; Probe: 5′-6FAM-AATGTCGTAACAACTCCG-MGB-3′) and eGFP assay (Forward: 5′-GGAGCGCACCATCTTCTTCA-3′; Reverse: 5′-CAGGGTGTCGCCCTCGA-3; Probe: 5′-6FAM-CTACAAGACCCGCGCCGAGGTG-MGB-3′). Each reaction was loaded into the sample well of an eight-well disposable cartridge (Bio-Rad) along with droplet generation oil (Bio-Rad), and droplets were generated in a droplet generator (Bio-Rad). Droplets were transferred to a 96-well PCR plate, sealed, and amplified to the end point (95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 56 °C for 1 min, and 72 °C for 15 s followed by a final 98 °C heat treatment for 10 min). The PCR plate was subsequently scanned on a Q × 100 droplet reader (Bio-Rad) and the data were analysed with QuantaSoft software (Bio-Rad).