User manual

6 | Droplet Digital
™
PCR Applications Guide
Droplet Digital
™
PCR
Fig. 1.10. 2-D plot of droplet fluorescence.
Droplet Digital PCR data from a duplex experiment in which two targets are PCR amplified
can also be viewed in a 2-D plot in which channel 1 fluorescence (FAM) is plotted against
channel 2 fluorescence (HEX or VIC) for each droplet (Figure 1.10).
Because the DNA distribution into the droplets follows a random pattern, droplets cluster
into four groups:
■
FAM negative, HEX negative (double-negative droplets)
■
FAM positive, HEX negative
■
FAM negative, HEX positive
■
FAM positive, HEX positive (double-positive droplets)
QuantaSoft
™
software measures the number of positive and negative droplets for each
fluorophore in each sample. The software then fits the fraction of positive droplets to a
Poisson algorithm to determine the starting concentration of the target DNA molecule in
units of copies/µl input (Figure 1.11).
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000
Channel 1 amplitude
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
Channel 2 amplitude
Fig. 1.11. Sample concentrations are plotted as copies/µl from the sample.
0 256 256 64 64 16 16 4 4 1 1
Sample
Concentration, copies/µl
10,000
1,000
100
10
1
0.10
0.01
1,390 1,400 1,400 1,400 1,360 1,370 1,380 1,390 1,370 1,360
260 259
64.2 64.8
15.5 15
4.63 4.68
1.46 1.53