User manual

Absolute Quantification and the Statistics of Droplet Digital
™
PCR
32 | Droplet Digital
™
PCR Applications Guide
Fig. 3.3. Outcome of ddPCR in two modes of analysis.
Low Concentration Example
When there are far fewer molecules than partitions (for example, 500 molecules or less in
20,000 partitions), it is relatively easy to see how the ddPCR approach enables accurate
quantification. Figure 3.3 shows a sample that contains six target DNA molecules.
If the entire sample is converted into droplets, we will expect to find exactly six droplets
that contain template, since it is statistically highly unlikely that a single droplet will start
with more than one target molecule.
For 20,000 droplets, the following concentration calculation is used (assumes an
approximate droplet volume of 1 nl or 10
–3
µl).
Volume analyzed = 20,000 x 10
–3
µl/droplet = 20 µl
Concentration = = = 0.3 copies/µl
Copies of target
Volume analyzed
6 copies
20 µl
Note: The final concentration is the same if we analyze 10,000 droplets and observe three
copies of target:
Volume analyzed = 10,000 x 10
–3
µl/droplet = 10 µl
Concentration = = = 0.3 copies/µl
Copies of target
Volume analyzed
3 copies
10 µl
6 copies of target in 20 µl sample
(0.3 copies/µl)
Most likely outcome of ddPCR:
6 positive droplets