User's Manual

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Chapter 4: Single DNA Molecule
Detection and Sorting of Droplets
Double emulsion droplets generated with the dPCR cartridge can be sorted and collected in a standard cell
sorter, capturing the DNA of interest. In this step, the positive dPCR droplets containing the region of interest
are identified and separated from the negative droplets using the fluorescent signal provided by the dPCR
amplification of the ID sequence (see Chapter 3).
Requirements for cell sorter
• A 488 nm (blue) laser.
• Optical configuration detecting fluorophores excited at 488 nm such as FITC, GFP, and PE.
• 100 μm nozzle tip/sorting chip minimum.
• Sample probe should be positioned at the bottom of the sample tube.
Notes to operator
• dPCR droplets are large, therefore the correct events are high in Forward Scatter (FSC) and Side
Scatter (SSC) (see Fig. 4.1).
• Smaller events and events with low SSC represent pure oil droplets and should be gated out.
• dPCR droplets are stable and relatively heavy. It can take up to 5-10 min depending on the sample
pressure before the dPCR droplets reach the point of interrogation and appear on the plot.
• Positive fluorescent droplets are likely to be very rare (possibly less than 0,02% of total dPCR
droplets). Therefore, a positive population can be difficult to identify. Make sure the live plot of
fluorescence shows at least 100.000 events (see Fig. 4.2) A histogram plot is not recommended.
• A threshold setting on FCS of about 5% or similar should be set to avoid disturbance by small
particles.
• dPCR droplets are relatively robust , set sample pressure to aim for a rate equal to 5000
events/sec.