Team:Northwestern/fluo

From 2013.igem.org

Fluorescence Assay

Grow an overnight culture in M9 Media
  • Aliquot 5 mL M9 media into an overnight tube
  • Add Antibiotics (5uL Amp 100)
  • Inoculate desired strain
  • Incubate and shake at 200rpm, 37 C
Grow the overnight culture in exponential phase
  • Aliquot 5 mL M9 media into an overnight tube
  • Add Antibiotics
  • Add 50 uL of overnight culture (shake gently to mix the cells that may have pelleted) into fresh media
  • Incubate and shake at 200 rpm, 37C
  • After 3 hours, test OD with cuvettes to check for exponential growth (usually around 0.3)
  • In 15 min intervals, check OD to make sure cells are in exponential phase
Prepare plate (should be done during the incubation time for growing exponential cells)
  • Add 175 uL of desired media (if testing with pH media, add it now)
  • Record which well contains what media
  • Make sure to work in triplicates
Plating exponential cells
  • Add 1 uL of exponential cell culture into desired well
  • Place in plate reader and test for both absorbance 600 and fluorescence 485
  • Test for 16 hours (time can be adjusted)