Lactococcus transformation
From OpenWetWare
| Line 1: | Line 1: | ||
| - | Preparing Electrocompetent Cells | + | ===Preparing Electrocompetent Cells=== |
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). | + | ::*(25ml M17 + 350µl glucose solution +5g Sucrose + 0.5g glycine). <br> |
| - | 3. Grow for ~4 hours until OD600 ~ 0.7. | + | 3. Grow for ~4 hours until OD600 ~ 0.7. <br> |
| - | 4. Chill culture on ice for 10 mins. | + | 4. Chill culture on ice for 10 mins. <br> |
| - | 4. Centrifuge cells for 15 mins at 3000g | + | 4. Centrifuge cells for 15 mins at 3000g. <br> |
| - | 5. Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol) | + | 5. Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br> |
| - | 6. Centrifuge cells for 15 mins at 3000g | + | 6. Centrifuge cells for 15 mins at 3000g. <br> |
| - | 7. Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol) | + | 7. Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br> |
| - | 6. Centrifuge cells for 15 mins at 3000g | + | 6. Centrifuge cells for 15 mins at 3000g. <br> |
| - | 7. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol) | + | 7. Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br> |
| - | 8. Separate into 100µl aliquots and store at -80°C until use. | + | 8. Separate into 100µl aliquots and store at -80°C until use. <br> |
| - | Electro-Transformation | + | ===Electro-Transformation=== |
| - | 9. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). | + | 9. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). <br> |
| - | 10. Add 900µl ice cold M17+ and keep on ice for 10 mins. | + | 10. Add 900µl ice cold M17+ and keep on ice for 10 mins. <br> |
| - | (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). <br> |
| - | 11. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. | + | 11. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br> |
| - | 12. Add entire subculture to GM17 with antibiotic. | + | 12. Add entire subculture to GM17 with antibiotic. <br> |
| - | 1ug/ml Erm for plates. | + | ::*1ug/ml Erm for plates. <br> |
| - | 5ug/ml Erm for culture. | + | ::*5ug/ml Erm for culture. |
[[Category:Protocol]] | [[Category:Protocol]] | ||
Revision as of 15:39, 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).
- (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.
