QIAcuity EG PCR Kit (25mL)

A 3x concentrated EvaGreen-based PCR master mix supplied by QIAGEN for singleplex quantification of DNA or cDNA targets on QIAcuity digital PCR instruments. The kit contains QuantiNova DNA Polymerase and a fluorescent reference dye for reliable detection of proper partition filling within dPCR Nanoplates; the EvaGreen signal is read in the FAM channel.

For optimal efficiency, amplicons should ideally be 60–150 bp in length. The recommended cycling protocol starts with a 2 min heat activation at 95°C, followed by 40 cycles of denaturation (15 s at 95°C), annealing (15 s at 55–62°C) and extension (15 s at 72°C), with a final 5 min cooling step at 40°C. Total reaction volume is 12 µl for the Nanoplate 8.5k (using 4 µl of master mix) or 40 µl for the Nanoplate 26k (using 13.3 µl of master mix). Genomic DNA samples ≥20 kb should be fragmented by restriction digestion at 0.025–0.25 U/µl for 10 min at room temperature before partitioning; restriction digestion is not required for highly fragmented DNA, FFPE DNA, circulating DNA, or cDNA.

Store at –30 to –15°C in a constant-temperature freezer, protected from light; the master mix may also be kept protected from light at 2–8°C. Components remain stable for 6 months under these conditions.