Protocol for cell lysis

Please click this hyperlink to access the virtual lab bench

Lab Safety - please contact your institution's health and safety office prior to implementing the laboratory procedure outlined. Comply with all laboratory safety regulations in accordance with your institution's health and safety office.

   

Materials and Reagents 

 
 Reagents:  Materials: 
  • An actively growing culture of BL21 (DE3) E. coli cells that have been transformed with your pET26-folA plasmid.
  • 25 units/mL benzonase nuclease
  • Protease inhibitors
    • 1 mM PMSF (phenylmethylsulfonyl fluoride)
    • 1 mM benzamidine
  • 50 mL polypropylene centrifuge tubes
  • Floor centrifuge and rotor
  • Room temperature shaker
  • 50 mL conical tubes

 

1. Add BugBuster TM protein extraction reagent at a 5/1 ratio (i.e. 5 mL of reagent for every 1 g of wet cell pellet). If your pellet weighs less than 1 gram, please add 5 mL of protein extraction reagent. 

2. Re-suspend the cell pellet in the protein extraction reagent (please ensure that your cell pellet is fully re-suspended in solution, not just detached off the side of the tube). You can re-suspend the pellet by pipetting it up and down in the solution added.

3. Incubate the re-suspended cells at room temperature for 20 minutes in the shaker at low speed.

4. Combine all re-suspended cell suspensions/ lab mentor into one, 50 mL polypropylene tube (choose any one tube from your lab mentor team). To pellet the cellular debris, spin the lysed cells at 16,000 x g for 30 minutes at 4 ˚C in the refrigerated floor centrifuge.

5. Each lab pair: remove your specific volume of supernatant from the combined tube and transfer the supernatant into one, 50 mL Conical tube. Make sure you label the tube appropriately.

6. Place cell lysate on ice. Proceed to the column chromatography step (in practice we ask our students to prepare the column while lysing cells).

 

License

Icon for the Creative Commons Attribution-NonCommercial 4.0 International License

BBS OER Lab Manual Copyright © 2021 by Felicia Vulcu/ Vivian Leong/ Caitlin Mullarkey (Department of Biochemistry and Biomedical Sciences McMaster University) is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.

Share This Book