Example 71. Appendix
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`Further aspects of
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`the present disclosure are described in
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`the attached
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`Appendix.
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`Example 2. Selected Embodiments
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`This example describes selected aspects and embodiments related to breaking
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`an emulsion of droplets, presented without
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`limitation as a series of numbered
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`paragraphs.
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`1.
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`A method of breaking an emulsion, comprising:
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`selecting an emulsion of aqueous droplets containing amplified nucleic acid;
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`10
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`mixing the emulsion with an organic solvent; and
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`centrifuging the emulsion mixed with the organic solvent.
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`2.
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`The method of paragraph 1, wherein aqueous droplets are encapsulated
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`by a proteinaceousskin.
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`3.
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`The method of paragraph 2, wherein the proteinaceous skin includes
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`15
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`serum albumin.
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`4.
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`The method of claim 1, wherein the step of mixing includes a step of
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`vortexing and/or shaking the emulsion in the presence of the organic solvent.
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`5.
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`The method of paragraph 1, further comprising a step of thermally cycling
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`the emulsion to promote nucleic acid amplification.
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`20
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`6.
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`The method of paragraph 1, wherein the organic solvent
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`includes a
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`compound that denatures protein.
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`7.
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`The method of paragraph 6, wherein the organic solvent
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`includes a
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`halogen-substituted hydrocarbon.
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`8.
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`The method of paragraph 7, wherein the halogen-substituted hydrocarbon
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`includes at least one chlorine.
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`9.
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`The method of paragraph 8, wherein the organic solvent
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`includes
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`chloroform.
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`10.
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`The method of paragraph 1, wherein the steps of mixing and centrifuging
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`form a continuous aqueous phase from the aqueous droplets, further comprising a step
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`of isolating at least a portion of the continuous aqueous phase from the organic solvent.
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`11.
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`The method of paragraph 10, further comprising a step of contacting the
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`continuous aqueous phase with a size-exclusion matrix to remove a residual amountof
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`10
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`the organic solvent.
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`12.
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`The method of paragraph 10, further comprising a step of precipitating
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`nucleic acid from the continuous aqueous phase.
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`13.
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`The method of paragraph 12, wherein the step of precipitating includes a
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`step of contacting the continuous aqueous phase with an alcohol, such as ethyl alcohol
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`15
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`or isopropyl alcohol.
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`14.
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`|The method of paragraph 1, wherein the emulsion includes a continuous
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`phase including a fluorinated oil.
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`15.
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`The method of paragraph 14, wherein the organic solvent is substantially
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`insoluble in the continuous phaseincluding a fluorinated oil.
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`20
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`16.
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`The method of paragraph 12, wherein the organic solvent is not miscible
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`with the continuous aqueous phase.
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`
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`17.
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`|The method of paragraph 1, wherein the steps of mixing and centrifuging
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`form a continuous aqueous phase, further comprising a step of determining a sequence
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`of nucleic acid obtained from the continuous aqueous phase.
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`18.
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`|The method of paragraph 17, wherein the step of determining a sequence
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`includes a step of performing a sequencing reaction on the nucleic acid obtained from
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`the continuous aqueous phase.
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`The disclosure set forth above may encompass multiple distinct inventions with
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`independent utility. Although each of
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`these inventions has been disclosed in its
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`preferred form(s), the specific embodiments thereof as disclosed and illustrated herein
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`10
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`are not to be consideredin a limiting sense, because numerous variations are possible.
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`The subject matter of the inventions includes all novel and nonobvious combinations
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`and subcombinations of the various elements, features, functions, and/or properties
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`disclosed herein.
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