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Team:UT-Tokyo/LabWork/AssayMethods
From 2012.igem.org
Assay Methods
Methods for Assay
H2 detection and assay
Gas Chromatography
Gas Chromatography is a method used for separating and analyzing compounds by injecting ａ sample mixture into a column prepared to be in a suitable stationary phase and passing a gas （carrier gas） in the mobile phase through the column, in order to separate the mixture into its components by making use of the difference in retention capacity against the stationary phase. This method can be applied to a gaseous or vaporizable sample, and is used for identification, purity testing, and quantitative determination.
Materials
- Gas Chromatograph ; SHIMADZU GC-8A with a thermal conductivity detector
Column ; molecular sieve 13X 60/80
Carrer gas ; nitrogen at 30 mL/min
Injection temperature ; 50℃
Column temperature ; 42℃
Current ； 90 mA
- 0.5 mL micro-syringe
- 2 mL vial
- Single colony of bacteria containing our construct that raises hydrogen production;
FhlA ; Plac－RBS－FhlA-d.term
m-FhlA ; Plac－RBS－(m-FhlA)－d.term
Protocol
1. A single colony of cells transformed with engineered plasmids (FhlA, m-FhlA) was inoculated into 2 mL of LB with appropriate antibiotics and grown to saturation at 37°C.
2. 100 μL of The saturated culture was added to fresh LB broth and grown till the OD600 = 0.4.
3. The culture was induced with 1 mM IPTG at 37°C for 1 hour. Then, formic acid was added so that its final concentration was 0 mM, 20 mM, or 60 mM.
4. 1mL LB broth was accurately added to a 2 mL vial. Then, gaseous phase substitution was carried out using nitrogen.
5. It was left to stand at 37℃ for 8 hours
6. 0.3 mL of the sample from the gaseous phase was injected into the gas chromatograph.
7. The peak area of the hydrogen obtained from gas chromatography was read, and the amount of hydrogen generated was calculated.
