Lactococcus transformation

Preparing Electrocompetent Cells

1. Grow cells overnight in 25ml of GM17 (350µl glucose solution to 25ml M17). 2. Add 1ml of overnight culture into 25ml SGM17 + 2.0% glycine (25ml M17 + 350µl glucose solution +5g Sucrose + 0.5g glycine). 3. Grow for ~4 hours until OD600 ~ 0.7. 4. Chill culture on ice for 10 mins. 4. Centrifuge cells for 15 mins at 3000g 5. Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol) 6. Centrifuge cells for 15 mins at 3000g 7. Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol) 6. Centrifuge cells for 15 mins at 3000g 7. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol) 8. Separate into 100µl aliquots and store at -80°C until use.

Electro-Transformation

9. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). 10. Add 900µl ice cold M17+ and keep on ice for 10 mins. (0.5M(.17g)Sucrose + 0.5%(15µl)Glucose+20mM(10µl)MgCl+0.2mM(10µl)CaCl) 11. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. 12. Add entire subculture to GM17 with antibiotic.

1ug/ml Erm for plates.

5ug/ml Erm for culture.