I have been trying to get a mutant marinum for almost 4 months now and nothing seems to work so far (not even a single colony appears on the plate). I would really be happy is you guys could share the marinum transformation protocol which works for you. I currently do the entire process of comeptent cell preparation and electroporation on the same day. I don't freeze the competent cells, I make them fresh every time. Below is the protocol which, I currently follow:

1. Culture 50 ml of Mycobacterium marinum culture in T-75 flasks and grow it standing in 32⁰C incubator for 3-4 days till it reaches a OD600 of 0.8-1

2. Chill culture on ice for 30 minutes with occasional shaking carry out all process in ice.

3. Transfer the cultures in 50 ml centrifuge tubes and pellet down the cells at 3.5K rpm for 10min at 4⁰C 

4. Suction off the supernatant and chill pellet on ice

5. Wash the pellet with 15-20ml ice cold sterile 10% glycerol 5-6 times

6. Resuspend the pellet in 1ml of cold 10% glycerol. 

7. Add 0.5-1ug of plasmid DNA to 400µl competent cells and transfer it to electroporation cuvette after mixing. Place everything on ice

8. Set electroporation apparatus: 2500V, 25µF, 1000Ω

9. Place the cuvette in holder and expose to one pulse (Biorad electroporator). A time constant of more than 16 should appear if not redo the entire process.

10. Quickly add the recovery media to the cuvette suspend the cells gently and transfer sample back to the test tube (600ul of 7H9+OADC media).

11. Incubate the cells standing at 32⁰C for 3-4 hours

12. Plate 100 µl cells on 7H11 plates containing appropriate antibody (I use hyg 80ug/ml) and pellet down rest of the cells and plate it on another plate.

13. Incubate the plates at 32⁰C until the colonies appear

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