Microfluidizer SOP

1.0 Safety
1.1 Understand the SOP before beginning
1.2 Wear proper safety attire, including: safety goggles, lab coats, and latex gloves.
1.3 The Microfluidizer runs at very high pressure so safety glasses must be worn at all times.

2.1 Sample prep

2.1 Re-suspend frozen cell pellets in 3:1 or 4:1 of cracking buffer until it forms a slurry.
2.2 The suspension of cells and buffer should remain below 10°C.

3.0 Microfluidizer prep

3.1 For >5L of sample to be cracked, connect the water supply and turn on the cold water.
3.2 Record the length of usage when you are done. (The oil needs to be changed at 50 hours).
3.3 Turn the air on and verify the pressure is at 60psi
3.4 Turn on the main power.
3.5 Surround the cracking chamber and cooling coil with ice and allow it to cool.

4.0 Priming the unit

4.1 Fill the reservoir with ~50mL of chilled buffer (without cells)
4.2 Turn the prime handle (located at the base of the reservoir) counter clockwise.
4.3 Turn the process pressure knob counter clockwise so it is set to minimum.
4.4 Pull the red intensifier button to start the flow of buffer.
4.5 Turn the process pressure knob clockwise to increase the pressure. Do not exceed 5000psi during
the prime step.
4.6 Once buffer comes out of the prime line, turn the prime handle clockwise to close the line.
4.7 Allow nearly all the remaining buffer to drain through the cracking chamber, checking for leaks.
If a leak occurs, stop and tighten as needed.
4.8 Press the intensifier button to stop flow once the buffer is below the tapered end of the reservoir.
If you let the buffer drain out of the reservoir you'll need to re-prime the unit.

5.0 Running the Microfluidizer

5.1 Verify the unit has been primed and your slurry is below 10C.
5.2 Pour the cell slurry into the reservoir.
5.3 Turn the process pressure knob counter clockwise so it is set to minimum.
5.4 Pull the red intensifier button to turn the start the flow of buffer.
5.5 Turn the process pressure knob clockwise to increase the pressure. Increase the pressure to the
desired level. E. coli will crack at 15,000 psi while yeast will need close to the maximum of
27,000 psi.
5.6 Collect the slurry into a container on ice.
5.7 Stop the unit once the cell/buffer mixture is below the tapered end of the reservoir. If you allow
the unit to run dry, you will need to prime it again.
5.8 Turn the process pressure knob counter clockwise so it is set to minimum
5.9 View the cell slurry under the microscope. It may take several passes through the cracking
chamber to disrupt the cells. ~95% of cells should be cracked for E. coli and ~65% for yeasts.
5.10 If needed, repeat 5.1 – 5.9 after the temperature is below 10°C.
5.11 Centrifuge the entire slurry at max speed for 30minutes – proceed with clean up.
5.12 After centrifugation, save the supernatant as it will be used for further processing.
 6.0 Clean Up
6.1 Fill the reservoir with ~500ml of Spore-Klenz.
6.2 Pull the red intensifier button to turn the start the flow of buffer.
6.3 Turn the process pressure knob clockwise to increase the pressure. Allow ~95% of the volume of
Spore-Klenz to flow through.
6.4 Fill the reservoir with ~1Lof 70% ethanol.
6.5 Turn the process pressure knob clockwise to increase the pressure. Stop the unit when ~750ml of
the ethanol solution has run through the system.
6.6 Turn the process pressure knob counter clockwise so it is set to minimum.
6.7 Cover the reservoir with parafilm and record the time used into the time log.