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Lactococcus transformation: Difference between revisions
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1. Grow cells overnight in 25ml of GM17 (M17 with 1% glucose). <br> | 1. Grow cells overnight in 25ml of GM17 (M17 with 1% glucose). <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> | ||
:*SGM17 = 25ml M17 + 0.25g glucose + 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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1. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). <br> | 1. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). <br> | ||
2. Add 900µl ice cold M17+ and keep on ice for 10 mins. <br> | 2. Add 900µl ice cold M17+ and keep on ice for 10 mins. <br> | ||
:*To eah ml of M17 add the following: 0.5M(.17g)Sucrose + 0.5%(15µl)Glucose + 20mM(10µl)MgCl<sub>2</sub> + 0.2mM(10µl)CaCl<sub>2</sub>. <br> | |||
3. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br> | 3. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br> | ||
4. Plate with appropriate antibiotic. <br> | 4. Plate with appropriate antibiotic. <br> | ||
