User manual

iii | Droplet Digital
™
PCR Applications Guide
Table of Contents
Chapter 1 Droplet Digital
™
PCR ............................................................1
Introduction ....................................................................1
QX100/QX200 Workflow ..........................................................2
Droplet Generation ............................................................3
PCR Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Droplet Reading ..............................................................4
ddPCR Data Analysis ..........................................................5
Emerging Applications of Droplet Digital PCR ..........................................7
ddPCR for Absolute Quantification and Experimental Considerations .........................7
Chapter 2 Designing Droplet Digital
™
PCR Experiments .......................................11
Assay Design for Droplet Digital PCR ................................................11
Designing Primers ...........................................................11
Designing Probes ............................................................12
Designing an Assay ..........................................................13
Sample Preparation .............................................................15
Adding DNA to the Reaction Mix ................................................16
ddPCR Experimental Workflow ....................................................17
Droplet Generation ...........................................................17
PCR ......................................................................18
Setting Up an Experiment in QuantaSoft
™
Software ..................................19
Droplet Reading .............................................................20
Data Analysis ...............................................................20
Merging Wells ...............................................................22
PCR Optimization Using Thermal Gradients ...........................................23
ddPCR Using the QX200 System and EvaGreen dsDNA Dye .............................24
EvaGreen and Gene Expression .................................................26
Multiplexing with EvaGreen .....................................................27
Reference ....................................................................27
Chapter 3 Absolute Quantification and the Statistics of Droplet Digital
™
PCR ..................... 28
Running Absolute Quantification Experiments .........................................28
Absolute Quantification Data Analysis .............................................29