Electroporation of 20ZR
Day 1:
- Inoculate 2-3 bottles (with 25ml P 0.75% media) with 20ZR
- 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:
- 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
- Centrifuge the 20ZR culture at 4700 rpm for 15 min at 4oC
- Remove and discard the supernatant without disturbing the pellet (NOTE: I decant the supernatant)
- Re-suspend the pellets in a total of 10 ml room temperature DI H2O (milliQ water) and transfer to 10.0 ml centrifuge tube.
- Centrifuge for 5 min at 4700 rpm at 4oC.
- Discard the supernatant without disturbing the pellet. Add 5 ml of DI H2O to the pellet but DO NOT suspend the pellet.
- Centrifuge for 5 min at 4700 rpm at 4oC.
- Discard the supernatant without disturbing the pellet. Wash the pellet with 5 ml DI H2O. DO NOT centrifuge discard the wash.
- 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
- Transfer 50ml of cell suspension into an eppendorf tube
- Add DNA (less than 6ml of DNA, ~300-500 ng DNA ) to appropriate tubes and mix gently
- Transfer to an ice-cold 0.1cm gap electroporation cuvette
- Electroporate with 1.5kV, 200 Ω, 25mF (time constant is usually 4.5-5ms) and immediately add 1ml room temperature P3% media.
- Transfer the electroporated cells into 10ml P3% in 125ml bottles.
- 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