APPENDIX
`
`
`
`Appendix
`Exemplary Droplet Breaking Protocol
`
`There exists a need to harvest amplification products reliably and efficiently from
`emulsion-based partitioning of reaction mixtures. Physical-based methods typically
`involve creating mechanical shear forces to rupture the water-in-oil emulsion and the
`emulsion stabilizing agent through multiple freeze-thaw cycles and/or centrifugation.
`Chemical methods utilized are dependent on the oil that is utilized to create the water-
`in-oil emulsion, and for silicone-based oils typically involve the use of a variety of
`organic solvents such as diethyl ether and ethyl acetate to remove the organic phase,
`coupled with precipitation to recover the desired product. This Appendix describes a
`method for breaking emulsions created using fluorinated hydrocarbons,
`in particular
`those created for PCR through the inclusion of a stabilization reagent.
`
`1. Following ddPCR, transfer droplets to 0.5 ml or 1.5 ml tubes (based on volume of
`droplets transferred).
`
`2. Add 1 volume of biotechnology grade chloroform (e.g., Sigma cat no. 288306)
`and vortex vigorously for 10 seconds.
`
`3. Add 1 volume of biotechnology grade chloroform (e.g., Sigma cat no. 288306)
`and vortex vigorously for 10 seconds
`
`
`
`Figure 1. Recovery of DNA from droplets following PCR.
`
`is mixed with an equal volume of chloroform,
`Panel A. 200 ul of droplets (i)
`causing the formation of a flocculate precipitate (ii). Subsequent centrifugation
`creates a large proteinaceous layer at the interface between the organic and
`aqueous phases.
`
`to centrifugation disrupts the flocculate
`Panel B. Vigorous vortexing prior
`material,
`resulting in a smaller layer at
`interface between the organic and
`aqueous phases, facilitating removal of the aqueous layer.
`
`4. Centrifuge for 10 min @ 18,000 xg.
`
`5. Carefully remove aqueous layer containing recovered PCR products without
`disturbing proteinaceousinterface
`
`Appendix
`
`Page 1
`
`
`
`6. Backextract the organic phase with 0.5 volumes of Tris-EDTA (TE) buffer and
`centrifuge as above.
`
`7. Pool recovered aqueous layers and buffer exchange thrice with TE buffer via
`ultramicrofiltration device (Millipore YM-30, NUWCO = 30 kDa) 15 min @ 8,000
`xg to remove contaminating reagents.
`
`8. Assess concentration and purity via UV-VIS spectrophotometry and gel analysis.
`Approximately 1 ug per 50 ul of droplets are expected, with a 260/280 ratio of
`>1.8.
`
`[he]
`
`Ladder
`
`Sample 1
`
`Sample?
`
`Sample3
`
`Sample 4
`
`SampleS
`
`Sampleé
`
`Sample?
`
`Sample
`
`Sample9
`
`Sample 10 Sample ii Sample 12
`
`FRIES Reiss ES SRRRS RRS RRS RRS
`TOES sees
`700) = aes
`
`apo ae eres
`7
`
`20g +
`L8G =
`100 ba
`BO
`{Ses
`
`
`
`L
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`*
`
`3
`
`9
`
`10
`
`11
`
`12
`
`foe]
`
`Eade)
`TOG
`S300
`
`= aod
`= 350
`
`= 200
`= 50
`ceo
`Pe
`25
`
`§
`X
`g
`SeamanYay
`Aqueous
`Aqueous
`layer
`layer
`Post-YM30
`clean-up
`
`Appendix
`
`Page 2
`
`
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