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Team:Hong Kong HKU/expressionoptimization
From 2014.igem.org
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BL21(DE3) cells carrying pETE were inoculated in 100ml LB supplemented with 50mg/ml Kanamycin. Cells were grown at 37°C until OD ~0.8, and were separated into fraction in Falcon tubes, into which IPTG was added to concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0mM. Cells were incubated overnight at room temperature, after which they were collected at 4,800g for 10min and washed once with sonication buffer*. Cells were pelleted after washing and a volume of 1ml/0.1g wet cell mass of sonication buffer were added. Sonication was performed with 2.0s bursts and 8.0s resting intervals for 3 min intervals on ice, between which time was allowed for the sonication mix to cool to ice temperature to avoid boiling of the liquid. After sonication was completed, the samples were clarified at 9,520g for 10min, the supernatant were withdrawn and total protein concentration assayed by the Bradford method. Normalization of the total protein concentration was carried by dilution in the sonication buffer, and protein samples were boiled for 10min in SDS-PAGE loading dye supplemented with 1% β-mercaptoethanol. Boiled samples was finally run on a 8% stacking gel/18% separating gel for analysis. The electrophoresis was carried out for 2h at 120V. The recovered gel was stained with Coosmassie blue stain for 1h and destained overnight as previously described. | BL21(DE3) cells carrying pETE were inoculated in 100ml LB supplemented with 50mg/ml Kanamycin. Cells were grown at 37°C until OD ~0.8, and were separated into fraction in Falcon tubes, into which IPTG was added to concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0mM. Cells were incubated overnight at room temperature, after which they were collected at 4,800g for 10min and washed once with sonication buffer*. Cells were pelleted after washing and a volume of 1ml/0.1g wet cell mass of sonication buffer were added. Sonication was performed with 2.0s bursts and 8.0s resting intervals for 3 min intervals on ice, between which time was allowed for the sonication mix to cool to ice temperature to avoid boiling of the liquid. After sonication was completed, the samples were clarified at 9,520g for 10min, the supernatant were withdrawn and total protein concentration assayed by the Bradford method. Normalization of the total protein concentration was carried by dilution in the sonication buffer, and protein samples were boiled for 10min in SDS-PAGE loading dye supplemented with 1% β-mercaptoethanol. Boiled samples was finally run on a 8% stacking gel/18% separating gel for analysis. The electrophoresis was carried out for 2h at 120V. The recovered gel was stained with Coosmassie blue stain for 1h and destained overnight as previously described. | ||
Latest revision as of 02:47, 18 October 2014
Expression Optimization
BL21(DE3) cells carrying pETE were inoculated in 100ml LB supplemented with 50mg/ml Kanamycin. Cells were grown at 37°C until OD ~0.8, and were separated into fraction in Falcon tubes, into which IPTG was added to concentrations of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0mM. Cells were incubated overnight at room temperature, after which they were collected at 4,800g for 10min and washed once with sonication buffer*. Cells were pelleted after washing and a volume of 1ml/0.1g wet cell mass of sonication buffer were added. Sonication was performed with 2.0s bursts and 8.0s resting intervals for 3 min intervals on ice, between which time was allowed for the sonication mix to cool to ice temperature to avoid boiling of the liquid. After sonication was completed, the samples were clarified at 9,520g for 10min, the supernatant were withdrawn and total protein concentration assayed by the Bradford method. Normalization of the total protein concentration was carried by dilution in the sonication buffer, and protein samples were boiled for 10min in SDS-PAGE loading dye supplemented with 1% β-mercaptoethanol. Boiled samples was finally run on a 8% stacking gel/18% separating gel for analysis. The electrophoresis was carried out for 2h at 120V. The recovered gel was stained with Coosmassie blue stain for 1h and destained overnight as previously described.
*Sonication buffer composition: 50mM Tris-HCl, pH 8.0
