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HpaC Assay

The theory behind this assay depends on the absorbance of NADH at 340 nm. HpaC produces FMNH2 from FMN in order to recycle reducing power. In the process, NADH is converted to NAD+. Because of this, the amount of NADH in solution will drop over time, which can be monitored by recording the absorbance at 340nm over time. Note: NADH and FMN stock solutions were aliquoted and stored in the -80°C freezer to prevent degradation.

  1. Grow up cultures of E. coli J04500-hpaC and J04500-dszB overnight at 37*C in LB with appropriate antibiotics.
  2. 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.
  3. 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).
  4. Freeze-thaw Cells (ethanol over dry ice, then 37*C waterbath) 5 times in order to lyse.
  5. Pipette different volumes of supernatant into a cuvette, and bring up to 998uL with Tris-HCl (pH 7.5).
  6. 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.