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Team:Calgary/Notebook/Protocols/hpac
From 2012.igem.org
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<li> The following morning, subculture 1 mL of each into 3 mL fresh LB with antibiotics, and add 200 uM IPTG to induce protein expression. Grow these cultures for 2h at 37*C.</li> | <li> The following morning, subculture 1 mL of each into 3 mL fresh LB with antibiotics, and add 200 uM IPTG to induce protein expression. Grow these cultures for 2h at 37*C.</li> | ||
<li>Take out 1 mL of cells, spin down to pellet. Discard supernatant, and wash 2x with 50 mM Tris-HCl (pH 7.5). Discard supernatant, and resuspend in 1 mL of 50 mM Tris-HCl (pH 7.5). </li> | <li>Take out 1 mL of cells, spin down to pellet. Discard supernatant, and wash 2x with 50 mM Tris-HCl (pH 7.5). Discard supernatant, and resuspend in 1 mL of 50 mM Tris-HCl (pH 7.5). </li> | ||
| - | <li>Freeze- | + | <li>Freeze-thaw Cells (ethanol over dry ice, then 37*C waterbath) 5 times in order to lyse.</li> |
<li>Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).</li> | <li>Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).</li> | ||
<li>Blank the spectrophotometer at 340nm with this sample, add 140uM NADH and 20uM FMN, invert quickly to mix, and read absorbance at 340 nm every 15s for 10 minutes. As a control, add 1 mL of Tris-HCl (pH 7.5), blank the spectrophotometer, add 140uM NADH and 20uM FMN, invert to mix, and read every 15s for 10 min.</li> | <li>Blank the spectrophotometer at 340nm with this sample, add 140uM NADH and 20uM FMN, invert quickly to mix, and read absorbance at 340 nm every 15s for 10 minutes. As a control, add 1 mL of Tris-HCl (pH 7.5), blank the spectrophotometer, add 140uM NADH and 20uM FMN, invert to mix, and read every 15s for 10 min.</li> | ||
Revision as of 02:20, 2 October 2012
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HpaC Assay
- Grow up cultures of E. coli J04500-hpaC and J04500-dszB overnight at 37*C in LB with appropriate antibiotics.
- The following morning, subculture 1 mL of each into 3 mL fresh LB with antibiotics, and add 200 uM IPTG to induce protein expression. Grow these cultures for 2h at 37*C.
- Take out 1 mL of cells, spin down to pellet. Discard supernatant, and wash 2x with 50 mM Tris-HCl (pH 7.5). Discard supernatant, and resuspend in 1 mL of 50 mM Tris-HCl (pH 7.5).
- Freeze-thaw Cells (ethanol over dry ice, then 37*C waterbath) 5 times in order to lyse.
- Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).
- Blank the spectrophotometer at 340nm with this sample, add 140uM NADH and 20uM FMN, invert quickly to mix, and read absorbance at 340 nm every 15s for 10 minutes. As a control, add 1 mL of Tris-HCl (pH 7.5), blank the spectrophotometer, add 140uM NADH and 20uM FMN, invert to mix, and read every 15s for 10 min.
