Protocols

Transformation of Listeria monocytogenes

Posted on Thursday, October 16, 2003

Description
A 2 days program to obtain tranformant Listeria

Procedure
1 - Preparation of competent Listeria monocytogenes
Day 1:
1. Prepare reagents needed:
- 2X BHI (37g BHI for a final volume of 500ml, autoclave for 15 minutes)
- 1 M sucrose (171.15g sucrose for a final volume of 500ml, filter sterilize)
- BHI-0.5M sucrose is prepared by mixing 2X BHI and 1M sucrose vol/vol
2. Start overnight culture of Listeria monocytogenes in 15 ml of BHI-0.5M sucrose

Day 2:
3. Subculture the overnight culture of Listeria monocytogenes (1/20) in 250 ml of BHI-0.5M sucrose
4. Grow to OD600=0.4 (˜4 hrs), then add 12.5 g/ml fresh penicillin G (1 ml from stock solution: 12.5 mg/ml PG + 20 ml 2N NaOH), to form pores in the membrane
5. Grow to OD600=0.7 (˜2.5 hrs), then place on ice for 10 minutes
6. Pellet Listeria monocytogenes at 10,000rpm for 5 mn, 4°C
7. Wash cells with ice-cold 0.5M sucrose, resuspending them by swirling in 200 ml.
8. Repeat 6-7 2 times, keeping cold
9. Resuspend the cell pellet in 2.5 ml of 0.5M sucrose (pasty solution)
10. Aliquot in 50-100 l samples (ready for electroporation), can be stored at -80°C

2 - Electroporation/transformation of competent Listeria monocytogenes
11. Mix 50 l competent Listeria monocytogenes with 1 g DNA (<5 l). Keep cold!
12. Pulse Listeria monocytogenes with 12.5 kV/cm, 25 …, 200 (˜5msec)
13. Immediately add 950 l BHI-0.5M sucrose and grow for 2 hrs, shaking, at 30°C
14. Plate Listeria monocytogenes on selective media at 30°C and wait at least 2 days for colonies.


Recipes


Supplies


Tips
Listeria monocytogenes is not E. coli, expect MUCH less colonies
Good luck

Submitted by: Mathilde