Periplasmic Lysis with Chloroform

Equipment & Consumables:

• Induced cells from Induction of Protein Expression

  • + Negative & Positive Control

• Micropipette and Sterile tips

• Sterile Workspace

• Vortex

• Freezer

• Centrifuge

• Ice in Ice Box

• 0.01M Tris Hydrochloride stock solution

• Chloroform (CHCl3)

• Microcentrifuge (Eppi) tubes

Protocol:

1. Thaw cells if they’ve been frozen.

2. Briefly vortex cells in the remaining medium.

3. Add 20 μl CHCl3 and briefly vortex.

4. Leave at room temperature for 15 minutes

5. Add 200 μl of 0.01M Tris Hydrochloride (pH 8.0)

6. Centrifuge 20 minutes x 6000g

   • Check your large centrifuge can reach this speed, otherwise transfer to small tube and use smaller centrifuge

7. Withdraw supernatant, store 20 μl in the freezer for SDS PAGE analysis and then proceed with the remaining supernatant to the Purification protocols.

   • The supernatant contains the released periplasmic material & hopefully your protein of interest if it was exported correctly.

Acknowledgements:

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC215841/