Chez Charles/ tags/ pages tagged emulsion

How to add contents to droplets ?

  • In Agresti et al., 2005, small molecules mixed with EtOH or DMSO were added by vortexing.
  • In Bernath et al. , 2005, nanodroplets were prepared with mineral oil, 7.5 % Span 80 and 2.5 % Tween 80, and incupated with the emulsion, with gentle mixing.

How to break the droplets ?

  • In Nakano et al. , 2005, by centrifugation (oil: silicone; surfactant: Triton X-100 0.1%).
  • In Agresti et al., 2005, by centrifugation in presence of diethyl ether (oil: mineral; surfactant: Triton X-100 0.1% and Span 80 4.5%).
  • High salt:
    • In Dielh et al. , 2006, by pipetting up and down in 100 mM NaCl, 1 % SDS, 1 % Triton X-100 and Tris (oil: mineral, surfactant: ABIL WE09).
    • In Kojima et al. , 2005, by mixing with 1M NaCl, 5 mM Tris-HCl pH 8.0, 0.5 mM EDTA (oil: mineral; surfactant: Sun Soft No. 818SK).
  • Extraction of beads with hexane:oil 1:1, in Levy et al. , 2005 (oil: mineral, surfactant: 5.5% Span 80, 0.5% Tween 80, 0.1 % Triton X-100).

Different kinds of surfactants:

  • Span 80 4.5 % and Tween 80 0.5 % (oil: mineral; primary paper: Tawfik et al. , 1998, followed by many others, sometimes adding Triton X-100 and changing the concentrations).
  • ABIL WE09 (polysiloxane–polycetyl–polyethylene glycol copolymer; primary paper: Dielh et al. , 2004)
  • Sun Soft No. 818SK (polyglycerol esters of intersesterified ricinoleic acid; primary paper: Kojima et al. , 2005)
  • DC 5225C Formulation Aid / DC 749 Fluid (Dow Chemical Co.) Margulies et al. , 2005
  • 3% fluorosurfactant (RAN Biotechnologies) in Novec HFE-7500 / 0.015% Tween 80 for double emulsions in Masted and coll., 2018.
  • 3% of Abil EM 180 or 4.5% Span 80/0.4% Tween 80/0.05% Triton X-100 were compared for ePCR by Terekhov and coll., 2020. “T-oil was less stable, displaying droplet coalescence after 25 cycles.” “ePCR clearly outperformed bulk PCR, reducing the number of reads with gross errors by twofold [and] resulted in a more uniform distribution of amplified sequences”

Reactive droplets:

  • Illumination changing magnetic or electric properties of droplets (Yang et al., 2018).
RSS Atom
Liquid drop of DNA libraries reveals total genome information.

Terekhov SS, Eliseev IE, Ovchinnikova LA, Kabilov MR, Prjibelski AD, Tupikin AE, Smirnov IV, Belogurov AA Jr, Severinov KV, Lomakin YA, Altman S, Gabibov AG.

Proc Natl Acad Sci U S A. 2020 Oct 21:202017138. doi:10.1073/pnas.2017138117.

Liquid drop of DNA libraries reveals total genome information.

“[...] either 3% of Abil EM 180 (A-oil) or a mixture of 4.5% Span 80/0.4% Tween 80/0.05% Triton X-100 (T-oil) was used [...] The homogeneity of the amplified DNA was much better in the case of A-oil, whereas ePCR in T-oil resulted in the formation of high-molecular-weight by-products [...] Thirty-five cycles of ePCR in A-oil were sufficient to reach saturation in the majority of the droplets. T-oil was less stable, displaying droplet coalescence after 25 cycles [...].” “Appearance of clear amplicon bands on electropherograms does not correlate with the uniformity of amplification during ePCR.” “ePCR clearly outperformed bulk PCR, reducing the number of reads with gross errors by twofold [and] resulted in a more uniform distribution of amplified sequences”

Posted
Xdrop: targeted sequencing of long DNA molecules from low input samples using droplet sorting

Esben Bjoern Madsen, Thomas Kvist, Ida Hoijer, Adam Ameur, Marie Just Mikkelsen

bioRxiv, September 6, 2018.

Xdrop: targeted sequencing of long DNA molecules from low input samples using droplet sorting

Enrichment by 1) encapsulating DNA fragments in double-emulsion water-in-oil-in-water droplets, 2) sorting the droplets containing the target sequence detected by a hydrolysis probe PCR, 3) extracting DNA from the droplets, 4) amplifying it by MDA (in a second generation of droplets), and finally 5) sequencing. When enriching for HPV DNA in HeLa genomes, 5 to 10% of the reads were on target. Emulsions were prepared in Dolomite chips.

Posted
Systems of mechanized and reactive droplets powered by multi-responsive surfactants.

Nature. 2018 Jan 18;553(7688):313-318. doi:10.1038/nature25137

Yang Z, Wei J, Sobolev YI, Grzybowski BA.

Systems of mechanized and reactive droplets powered by multi-responsive surfactants.

New amphiphilic surfactants containing gold, iron oxide or lead sulfide were used to create droplets that aggregate in magnetic fields, translate or rotate under laser illumination, and weld together electric fields. Combining these three manipulations allowed for the creation of complex structures used as microreactors for chemical reactions between reagents brought by individual droplets.

Posted
Directed evolution of polymerase function by compartmentalized self-replication.

Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4552-7 doi:10.1073/pnas.071052198

Ghadessy FJ, Ong JL, Holliger P.

Directed evolution of polymerase function by compartmentalized self-replication.

No beads. A bacterial cel is used as a subcompartment maintaining gene and proteins together. Evidence for leakage of small molecules at high compartment density.

Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.

Agresti JJ, Kelly BT, Jäschke A, Griffiths AD.

Proc Natl Acad Sci U S A. 2005 Nov 8;102(45):16170-5 doi:10.1073/pnas.0503733102

Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.

Small molecules mixed with EtOH or DMSO were added by vortexing.

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.

Wang BL, Ghaderi A, Zhou H, Agresti J, Weitz DA, Fink GR, Stephanopoulos G.

Nat Biotechnol. 2014 May;32(5):473-8. doi: 10.1038/nbt.2857

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption.

Screening genomic clones detected a tandem expansion of the XYLA gene.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

Anal Chem. 2012 Apr 17;84(8):3599-606.

Zhang H, Jenkins G, Zou Y, Zhu Z, Yang CJ.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

Primers bound to agarose by 5′ acrydite residues. RT and PCR chained in the same reaction mixture using hot start PCR enzyme. Agarose beads sorted by FACS.

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.

Nucleic Acids Res. 2005 Oct 6;33(17):e150 doi:10.1093/nar/gni143

Kojima T, Takei Y, Ohtsuka M, Kawarasaki Y, Yamane T, Nakano H.

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.

The composition of the mixture has been modified so that it stays stable in a PCR of 55 cycles of 10 minutes. Uses Sun Soft N° 818SK.

Posted
A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system.

Protein Eng Des Sel. 2004 Mar;17(3):201-4 doi:10.1093/protein/gzh025

Ghadessy FJ, Holliger P.

A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system.

Primary paper for the use of ABIL EM90. Span/Tween surfactants oxydise the reagents. DTT partly prevents oxydisation but inhibits reactions above 20 mM.

Posted
Genome sequencing in microfabricated high-density picolitre reactors.

Nature. 2005 Sep 15;437(7057):376-80. doi:10.1038/nature03959

Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM.

Genome sequencing in microfabricated high-density picolitre reactors.

Primary reference for “454” sequencing.