User manual

22 | Droplet Digital
™
PCR Applications Guide
Fig. 2.8. Setting thresholds in a 2-D plot.
Fig. 2.9. Merged well data.
For correct quantification of a duplex experiment, use the thresholding tools to ensure
correct designation of the droplet populations as double negative (gray), FAM positive (blue),
VIC/HEX positive (green), and double positive (brown = positive for FAM and HEX/VIC in the
same droplet). There is an autoanalysis tool as well as several different manual thresholding
tools including a “free-draw” tool. Once you have selected the desired color tab and draw
tool, click on the chart and select the droplets. They will change color when you are done.
Merging Wells
Selecting the merged button in QuantaSoft software combines the positive and negative
droplets from all the wells with the same name and analyzes these data together as a
single experiment. You can display merged and single wells simultaneously or individually.
All merged-well data are displayed with a closed symbol (Figure 2.9). In contrast, single-well
data are displayed with an open symbol (see Figure 1.11).
256 64 16 4 1
Sample
Concentration, copies/µl
10,000
1,000
100
10
1
0.10
0.01
1,400 1,400 1,360 1,390 1,360
259
64.5
15.2
4.65
1.5
Designing Droplet Digital
™
PCR Experiments