Figure 3.

Metering requires calibration but is highly reproducible. The peristaltic pump (6) meters discrete amounts of fluid into the ring every time it is cycled (21). (A) To calibrate metering volumes against the number of pump cycles, fluorophore in the reaction ring (cartoon, blue) was displaced by metering a variable number of pump cycles of buffer (cartoon, grey) into the ring. The remaining fluorophore was mixed with buffer, and quantified by measuring fluorescence. Eight pump cycles (orange circle) displace (1–0.58)(5 nl) = 2.1 nl, where 5 nl is the calculated ring volume. (B) Repeatedly metering and mixing the same amount of buffer creates a serial dilution. Here, eight pump cycles serially dilute the sample by a factor of 1/0.58 = 1.7 per cycle. As serial dilutions would amplify metering error, the similar curves demonstrate a high degree of reproducibility.