Lactococcus transformation
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m (Preparation of electro-competent Lactococcus lactis moved to Transforming Lactococcus lactis: better description) |
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| - | + | Preparing Electrocompetent Cells | |
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| + | 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. | ||
| + | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
Revision as of 15:32, 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). 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.
