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The flask was then put into a water bath at 60 °C and the polymerization mixture was flushed with nitrogen gas for 30 min, and a mechanical stirrer to exclude oxygen from the system.
Required amount of cycloheptatriene and substituted aniline dissolved in 2 mL N-methyl pyrrolidine were added and the reaction mixture was flushed with argon for 5 min, then it was stirred at 140 °C for 72 h.
For each extraction, the mixture was flushed with nitrogen gas and shaken (300 rpm) for 2 h at room temperature.
After addition of DTT to a final concentration of 50 mM, the mixture was flushed with N2 and incubated for 3 h at 37°C.
The mixture was flushed with CO2, and approximately 10 mL of medium was transferred into 50 mL serum bottles under anaerobic conditions.
The resulting mixture was flushed with H2 three times and then stirred under H2 (1 atm, 101 kPa) at 50 °C overnight.
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Then, the aqueous laccase mixture suspension was flushed over the silica crystals.
The catheter in the jugular vein was flushed with a mixture of heparin and NaCl to avoid clotting.
Then, a mixture of pure nitrogen and Ar was flushed in as reaction gas with a pressure of 105 Pa.
Subsequently the medium was flushed with a mixture of nitrogen and CO2 (80 : 20 v/v) for 5 minutes and inoculated with 4 mL inoculum (10% v/v inoculum), before being incubated at 37°C with continuous stirring (150 rpm).
For hypoxic cell cultures, cells were placed in an airtight chamber that was flushed with a gas mixture of 5% CO2 and 95% N2.
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