User manual

Absolute Quantification and the Statistics of Droplet Digital
™
PCR
34 | Droplet Digital
™
PCR Applications Guide
Even at an average of 5 copies/droplet (the upper end of the recommended loading range),
we expect to see about 134 empty droplets in a total of 20,000 droplets (data not shown).
Looking across the Whole Concentration Range
Figure 3.4 shows the number of droplets with 0, 1, 2, 3 (and so on) copies of the target
DNA at different DNA concentrations. There is a simple mathematical relationship between
the fraction of droplets that are unoccupied (black bar) and the concentration of target
molecules. As the concentration of input target DNA increases, the expected number
of unoccupied droplets decreases.
Fig. 3.4. Number of droplets with copies of target DNA at different DNA concentrations.
CPD, copies per droplet.
Number of droplets
15,000
10,000
5,000
0
0.25 CPD 1 CPD 2.5 CPD
0 1 2 3 4
Number of target molecules in droplet
4,425 positives
15,576 negatives
0 1 2 3 4 5 6 7
12,642 positives
7,3 5 8 negatives
0 1 2 3 4 5 6 7 8 9
18,357 positives
1,642 negatives
Number of droplets
15,000
10,000
5,000
0
Number of droplets
15,000
10,000
5,000
0
Note: Quantification is impossible when there are no unoccupied droplets,
which is approached at 8 CPD.
Concentration Calculation
Definitions
C = copies per droplet (CPD)
E = observed fraction of empty droplets
V
droplet
= volume of droplet
N
neg
= number of negative droplets
N = total number of droplets