Lactococcus transformation: Difference between revisions
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1. Grow cells overnight in 25ml of GM17 (350µl glucose solution to 25ml M17).<br> | 1. Grow cells overnight in 25ml of GM17 (350µl glucose solution to 25ml M17).<br> | ||
2. Add 1ml of overnight culture into 25ml SGM17 + 2.0% glycine. <br> | 2. Add 1ml of overnight culture into 25ml SGM17 + 2.0% glycine. <br> | ||
::*(25ml M17 + 350µl glucose solution +5g Sucrose + 0.5g glycine). <br> | |||
3. Grow for ~4 hours until OD600 ~ 0.7. <br> | 3. Grow for ~4 hours until OD600 ~ 0.7. <br> | ||
4. Chill culture on ice for 10 mins. <br> | 4. Chill culture on ice for 10 mins. <br> | ||
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11. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br> | 11. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br> | ||
12. Add entire subculture to GM17 with antibiotic. <br> | 12. Add entire subculture to GM17 with antibiotic. <br> | ||
::*1ug/ml Erm for plates. <br> | |||
::*5ug/ml Erm for culture. | ::*5ug/ml Erm for culture. | ||
Revision as of 12:40, 5 April 2011
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).
- (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).
- (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.
- 1ug/ml Erm for plates.
