User manual

Droplet Digital
™
PCR Applications Guide | 35
Absolute Quantification and the Statistics of Droplet Digital
™
PCR
Formula for Calculating Concentration
The formula used by QuantaSoft software to calculate concentration is:
Derivation of Concentration Formula
The Poisson distribution gives probability Pr(n) that a droplet will contain n copies of target
if the mean number of target copies per droplet is C:
Inputting n = 0 gives the probability that a droplet will be empty for a given value of C:
Pr(0) = e
–c
For a large number of droplets, the observed fraction of empty droplets (E) gives a very
good estimate of Pr(0), so:
E = e
–c
Solving for C gives:
C = –In(E)
Recall that C is copies per droplet. To convert to copies per µl, divide by the droplet volume:
Combining the two previous equations gives:
Note that by definition:
Combining the two equations above gives:
Concentration =
–In(E)
V
droplet
E =
N
neg
N
Concentration = –In ( ) /V
droplet
N
neg
N
C = –In ( ) /V
droplet
N
neg
N
Concentration = –In ( ) /V
droplet
N
neg
N
Concentration =
C
V
droplet
Pr(n) =
C
n
e
–c
n!