Lactococcus transformation

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Current revision (14:06, 6 April 2011) (view source)
 

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Preparing Electrocompetent Cells

1. Grow cells overnight in 25ml of GM17 (M17 with 1% glucose).
2. Add 1ml of overnight culture into 25ml SGM17 + 2.0% glycine.

  • SGM17 = 25ml M17 + 0.25g glucose + 5g Sucrose + 0.5g glycine).

3. Grow for ~4 hours until OD600 ~ 0.7.
4. Chill culture on ice for 10 mins.
5. Centrifuge cells for 15 mins at 3000g.
6. Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol).
7. Centrifuge cells for 15 mins at 3000g.
8. Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol).
9. Centrifuge cells for 15 mins at 3000g.
10. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol).
11. Separate into 100µl aliquots and store at -80°C until use.

Electro-Transformation

1. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes).
2. Add 900µl ice cold M17+ and keep on ice for 10 mins.

  • To eah ml of M17 add the following: 0.5M(.17g)Sucrose + 0.5%(15µl)Glucose + 20mM(10µl)MgCl2 + 0.2mM(10µl)CaCl2.

3. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours.
4. Plate with appropriate antibiotic.

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