Protocol 9: Electroporation of 20 ZR

Electroporation of 20ZR

Day 1: 

  1. Inoculate 2-3 bottles (with 25ml P 0.75% media) with 20ZR
  2. Add methane (~40-45cc) to the bottles and incubate at 30oC until the OD is between 0.3 and 0.6 at 600 nm

Day 2: 

  1. Once the OD is between 0.3 and 0.6 at 600 nm, pool the three 20ZR cultures and divide them into two 50.0 ml centrifuge tubes 
  2. Centrifuge the 20ZR culture at 4700 rpm for 15 min at 4oC
  3. Remove and discard the supernatant without disturbing the pellet (NOTE: I decant the supernatant) 
  4. Re-suspend the pellets in a total of 10 ml room temperature DI H2O (milliQ water) and transfer to 10.0 ml centrifuge tube. 
  5. Centrifuge for 5 min at 4700 rpm at 4oC.
  6. Discard the supernatant without disturbing the pellet. Add 5 ml of DI H2O to the pellet but DO NOT suspend the pellet. 
  7. Centrifuge for 5 min at 4700 rpm at 4oC.
  8. Discard the supernatant without disturbing the pellet. Wash the pellet with 5 ml DI H2O. DO NOT centrifuge discard the wash. 
  9. Re-suspend the pellet in 100-150ml DI water (you will need 50ml/transformation) and place on ice. **From this point on, everything should be kept on ice until the electroporation is completed 
  10. Transfer 50ml of cell suspension into an eppendorf tube 
  11. Add DNA (less than 6ml of DNA, ~300-500 ng DNA ) to appropriate tubes and mix gently 
  12. Transfer to an ice-cold 0.1cm gap electroporation cuvette 
  13. Electroporate with 1.5kV, 200 Ω, 25mF (time constant is usually 4.5-5ms) and immediately add 1ml room temperature P3% media. 
  14. Transfer the electroporated cells into 10ml P3% in 125ml bottles.
  15. Add 40 cc methane and incubate the cultures at 30oC shaking incubator for overnight (~14-16 hrs). 

Day 3: 

· Centrifuge cells for 15 min at room temperature at 4700 rpm 

· Re-suspend cells in 500ml-1ml P3% media  

· Plate each suspension on 3-4 selective plates with appropriate antibiotics