User manual

Droplet Digital
™
PCR Tips, Assay Considerations, and Troubleshooting
84 | Droplet Digital
™
PCR Applications Guide
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Do not exceed the recommended DNA load (66 ng/well undigested DNA or
1,500 ng/well digested DNA)
■
Use only approved plates (Eppendorf twin.tec semi-skirted 96-well plates, catalog
#951020362) with approved pierceable foil heat seals (Bio-Rad catalog #181-4040)
■
Properly seal the 96-well plate. Under- or over-sealed plates result in oil evaporation
during thermal cycling and compromise droplet data quality. If using the PX1
™
PCR plate
sealer (Bio-Rad catalog #181–4000), seal plates at 180°C for 5 sec. Do not use the PX1
sealing protocol twice on the same plate because this often disrupts the original seal
■
Ensure that the full volume of the generated droplets is transferred into the 96-well plate
by inspecting the DG8 cartridge after transfer
■
Use only approved pipet tips for droplet generation and droplet transfer. Rainin and
Eppendorf tips are approved for use
For sample loading, use P-20 pipet tips and slowly dispense the sample into the bottom of
the DG8 well rather than pipetting at the top edge of the well. Then dispense 70 µl of oil into
the oil wells. Begin droplet generation within 2 min of oil loading.
Use a manual P-50 pipet with a normal bore P-200 tip (not wide or narrow bore) to transfer
droplets. Angle the P-200 tip in the well to prevent the droplets from having to squeeze
between the pipet tip and well bottom (angle the tip position such that it is not vertical in the
well). Slowly draw 40 µl of droplets into the pipet tip over ~5 sec. Typically ~5 µl of air will be
pulled into the tip, which helps prevent the oil from leaking out.
Position the pipet tip (containing the droplets) near the bottom of the well and dispense the
sample, ensuring ample room between the well and the pipet tip so that the droplets do not
shear upon dispensing.
Inconsistent Concentration Results
Technical replicates of the same sample should yield concentration estimates that are within
the Poisson confidence error bars 95% of the time. If the concentration estimates between
technical replicates are not close, the most common causes are poorly mixed reaction
mixtures or poor thermal cycler temperature uniformity.
Insufficient Mixing
When creating technical replicates, thoroughly mix the reaction mixture (master mix, sample,
and assay) by pipetting the reaction mixture up and down ten times, using 90% volume
strokes. Alternatively, pulse vortex the reaction mixture for 15 sec followed by spinning the
sample down. Do not assemble or mix reaction mixtures in the DG8 cartridge. Figure 9.6
shows replicates that were not sufficiently mixed before droplet generation.