Beadbeating Cell Lysis

Equipment & Consumables:

• Induced cells from Induction of Protein Expression

  • + Negative & Positive Control

• Micropipette and Sterile tips

• Sterile Workspace

• Bead-beater + sterile bead-beater tubes with glass beads inside

  • 1 large bead, small scoop of medium + small beads. Autoclave.

• Vortex

• Freezer

• Centrifuge (ideally refrigerated)

• Ice in Ice Box

• 10 mg/ml DNAse

• 10 mg/ml Lysozyme

• Wash Buffer

  • 50 mM sodium phosphate (pH8.0)

  • 300 mM NaCl

  • 10 mM imidazole (autoclaved)

• 15 ml Falcon tubes

• Microcentrifuge (Eppi) tubes

Protocol:

1. Resuspend each cell pellet in 2.5 mL Wash Buffer using the vortex and combine into one Falcon tube (total volume ~ 5 mL).

2. Add 100 µl DNAse (10 mg/ml)

3. Add 50-500 µl Lysozyme (10 mg/ml), final concentration 0.1-1 mg/ml.

4. Tap to mix and then freeze at -20°C for 30 minutes

5. Thaw cells in your hands and then allow ~10 minutes at Room Temperature to allow for lysozyme action.

6. Store tubes on ice.

7. Label sterile bead beater tubes (1, 2, 3, 4, 5,...., n) where n = (number of cultures x 5)

8. Distribute 1 ml of cell paste from one of your cultures into 5 sterile bead beater tubes. Keep chilled on ice.

   • Be very careful to not mix different cultures at this stage.

   • If you're working with more than one culture, do one at at a time, and use a table to keep track.

   • Labels come off very easily during beadbeating.

9. Perform the following beadbeater cycle for your 5 x tubes of cell paste and glass beads:

   1. Squeeze one beadbeater tube into the beadbeater, close the lid and then beat for thirty seconds.

   2. Pry loose the tube and return the tube to the ice, it will be noticeably warm.

   3. Repeat for the next tube.

   4. Continue until all tubes have gone through 3 x 30 second cycles of beating.

      • Note: This is time consuming, if you have to beadbeat multiple cultures, allocate plenty of time for this step.

10. Centrifuge bead beater tubes in the cold for 5 minutes at ~10,000 g to pellet beads and cell debris.

    • You may need a blank bead beater tube with some water as a balance in the centrifuge.

    • You also may need to put a centrifuge in the fridge/cold room for this step if you don’t have a refrigerated centrifuge.

11. Label a 15 mL Falcon Tube with (plasmid-cell-incubation time-incubation temp-name-date)

12. Combine supernatants into the 15mL Falcon tube

    • The insulin protein will be in the supernatant.

13. Take a 100 µl aliquot of crude lysate and add to a microcentrifuge tube labelled (plasmid-cell-crude lysate-incubation time-incubation temp-name-date) for downstream SDS page analysis.

14. You can freeze these tubes and continue tomorrow or proceed to the Centrifuge & Filter Purification protocol.

Acknowledgements:

• Coleman Protocols 2017 + 2019 http://coleman-lab.org/

• https://openwetware.org/wiki/Qiagen_Buffers