Avestin Emulsiflex S.O.P.

Stuff to bring

  • Extra lysis buffer (~30 ml)
  • Collection tube
  • Sample
  • Ice
  • Set up
    • Take cooling coil out of the beaker and add ice and water
    • Place coil in ice bath
    • Check that pressure gauge sitting on top of the machine reads 60-80 psi.
    • Take top off of intake funnel
    • Notes:
      • Resuspend sample in 2-4mL of lysis buffer per 1 g of cells
      • Sample will heat ~1°C per 1,000 psi used
      • To lyse E. coli, use 15-18,000 psi
        • Can use 25,000 but won’t improve lysis much and will heat the sample
      • To lyse yeast, use 26-28,000 psi
        • Can add lysozyme to chew up cell walls
      • There are two pressure gauges on the front of the machine
        • The small one to the left is the applied pressure (only gets up to ~80 psi
        • The large one to the right is what the cells are experiencing
          • This one will “jump” between 0 psi and the pressure through the machine. Watch the needle to make sure you are getting the right final pressure
        • Loading
          • The machine is stored in isopropanol
          • Turn and pull the stop button and press start
          • While keeping the applied pressure at 0 psi:
            • Allow isopropanol to run dry (into waste beaker)
            • Rinse intake funnel with water a few times and run dry
            • Rinse with lysis buffer and run dry
            • Hold the out flow tube over the intake funnel.
            • Pour sample in and allow sample to flow back into funnel
            • The instrument dislikes clumps! Your sample should be a homogeneous liquid – mix on a magnetic stir plate if needed. If you suspect there are clumps in your sample use the mesh sieve to remove clumps – otherwise the machine may get clogged.
  • Lysis
    • While keeping outflow tube above the funnel:
      • Quickly increase the applied pressure to 30 psi (using small white knob near the green start button)
      • Then slowly increase the pressure by turning the knob 1/3 turn at a time. You will soon see a momentary pause in the flow of liquid out of the tubing at which point pressure building in the homogenizing valve will be indicated on the large pressure gauge to the right.
      • Increase pressure by continuing to turn the grey/white knob.
      • Adjust the pressure as needed to maintain 15-18K psi.
      • Once the sample runs dry, the pump will stop
      • Turn the pressure back to 0 psi to allow remaining sample to flow out
    • Clean up
      • Take the funnel off and rinse it in the sink. Use soap and bottle brush to clean it.
      • Put funnel back on
      • Add water and run dry
      • Add ~20mL of 70% isopropanol
      • Press stop when you can tell isopropanol is being ejected.
      • Make sure there is still some left in the funnel to ensure the entire system is stored in isopropanol.
      • Cap the funnel