Frozen stocks of E. coli (ATCC 25922, ATCC BAA 2471) were thawed, washed twice, and diluted to 107 CFU/mL in Muller-Hinton II cation adjusted broth (Sigma-Aldrich). Separately, 400 μM resazurin (Sigma-Aldrich) was mixed with either 0 μg/mL or 8 μg/mL gentamicin (Sigma-Aldrich) in Mueller-Hinton broth. Both bacterial sample and resazurin/antibiotic solution were then drawn into separate 1-m-long sections of Tygon tubing (Cole-Parmer) with an inner diameter of around 500 μm. Both Tygon tubing sections were individually connected to Hamilton 1000 glass syringes (Sigma-Aldrich) containing FC-40 oil (Sigma-Aldrich), which served as the displacement fluid for injecting both aqueous samples from Tygon tubings into the device using a syringe pump at 15 μL/h (Harvard Apparatus). An oil phase consisting of FC-40 oil and 5% poly(ethylene glycol) di-(krytox-FSH amide) surfactant by weight was pumped into the device through the oil inlet of the device at 60 μL/h by a separate syringe pump. To confirm stable droplet generation, the device was imaged using a 4× objective lens and a CCD camera during droplet generation and after droplet incubation. Droplet incubation was conducted on chip at 37 °C using a controllable peltier heater on which the microfluidic device rested.
Fc 40 oil
Manufactured by Merck Group
FC-40 oil is a fluorinated hydrocarbon fluid produced by Merck Group. It is a clear, colorless, and odorless liquid. FC-40 oil is primarily used as a heat transfer fluid in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Resazurin, by Merck Group (1 mentions)
Tygon tubing, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Tygon tubing, by Cole-Parmer (1 mentions)
Polyethylene glycol 6000, by Merck Group (1 mentions)
Phix174 virion dna, by New England Biolabs (1 mentions)
T100 thermocycler, by Bio-Rad (1 mentions)
Platinum multiplex pcr master mix, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
4 protocols using fc 40 oil
1
Microfluidic Antimicrobial Susceptibility Assay
2
High-throughput co-encapsulation of methanotrophs and GESS
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For high-throughput co-encapsulation of methanotrophs and GESS, we used the μEncapsulator 1–2 Reagent Droplet Chip (50 μm, fluorophilic, Dolomite Microfluidics, Royston, United Kingdom). Monodisperse droplets with diameters of 40–50 μm were produced at the flow focusing junction, which has two aqueous inlets, two oil inlets, and an outlet channel. This chip interface interacts directly with the Sample Reservoir Chip (Dolomite Microfluidics), where the methanotroph sample and GESS/benzene solution were loaded through separate channels. Both channels were connected to a single pressure pump through PTFE tubing (Kinesis, Berlin Township, NJ, United States) with a Y-connector. The tubing sections were filled with FC-40 oil (Sigma-Aldrich), which served as the displacement fluid. By precisely controlling the pressure pump, a pressure of 300 mbar was used for aqueous sample delivery. An oil phase consisting of FC-40 with 2% Pico-Surf (Dolomite Microfluidics) was delivered through a single port of the microfluidic chip using a pressure pump at 500 mbar, and the flow was split equally into the two channels. Pico-Surf, a fluorinated surfactant, was added to stabilize the droplets.
3
Droplet-based Quantitative PCR Protocol
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PhiX-174 virion DNA (New England Biolabs) is mixed with PCR reagents containing 1X Platinum Multiplex PCR Master Mix (Life Technologies), 200 nM probe (IDT), 1 μM forward primer (IDT), 1 μM reverse primer (IDT), 0.5% (v/v) Tween 20 (Sigma-Aldrich), and 2.5% (w/v) Poly(ethylene glycol) 6000 (Sigma-Aldrich). The reaction mix is printed with a PDC70 capillary into 100 μL HFE 7500 oil with 5% (w/v) PEG-PFPE amphiphilic block copolymer in a 0.2 mL PCR tube. After printing, the oil is replaced with 50 μL FC-40 oil (Sigma-Aldrich) with 5% (w/v) PEG-PFPE amphiphilic block copolymer. The emulsion is amplified using the following program on a Bio-Rad T100 thermocycler: 2 m 30 s at 95 °C; 35 cycles of 30 s at 95 °C, 1 m 30 s at 60 °C, and 30 s at 72 °C; and a final extension of 5 m at 72 °C. The emulsion after thermocycling is imaged on the EVOS Cell Imaging System in brightfield and GFP channels. Intensity data is extracted from each droplet; coalesced droplets with a diameter greater than 80 μm were excluded from analysis. The Poisson estimator was calculated from the observed fraction of positive droplets by the following equation: 23 (link).
4
Droplet Generation and Separation Protocol
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