Lactococcus transformation: Difference between revisions
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===Preparing Electrocompetent Cells=== | ===Preparing Electrocompetent Cells=== | ||
1. Grow cells overnight in 25ml of GM17 ( | 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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10. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br> | 10. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br> | ||
11. Separate into 100µl aliquots and store at -80°C until use. <br> <br> | 11. Separate into 100µl aliquots and store at -80°C until use. <br> <br> | ||
===Electro-Transformation=== | ===Electro-Transformation=== | ||
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> | |||
:: | :*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> | |||
4. Plate with appropriate antibiotic. <br> | |||
[[Category:Protocol]] | [[Category:Protocol]] | ||
Latest revision as of 12:06, 6 April 2011
Back to Protocols
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).
- 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.
- 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.
