Lactococcus transformation

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Back to [[Protocols]]
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===Preparing Electrocompetent Cells===
===Preparing Electrocompetent Cells===
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1.  Grow cells overnight in 25ml of GM17 (350µl glucose solution to 25ml M17).<br>
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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>
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::*(25ml M17 + 350µl glucose solution +5g Sucrose + 0.5g glycine). <br>
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:*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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4.  Centrifuge cells for 15 mins at 3000g. <br>
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5.  Centrifuge cells for 15 mins at 3000g. <br>
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5.  Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
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6.  Gently shake to resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
-
6.  Centrifuge cells for 15 mins at 3000g. <br>
+
7.  Centrifuge cells for 15 mins at 3000g. <br>
-
7.  Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
+
8.  Resuspend pellet in 3ml Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
-
6.  Centrifuge cells for 15 mins at 3000g. <br>
+
9.  Centrifuge cells for 15 mins at 3000g. <br>
-
7.  Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
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10.  Resuspend pellet in 500µl Electroporation Buffer (0.5M Sucrose, 10% glycerol). <br>
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8.  Separate into 100µl aliquots and store at -80°C until use. <br> <br>
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11.  Separate into 100µl aliquots and store at -80°C until use. <br> <br>
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===Electro-Transformation===
===Electro-Transformation===
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9.  Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). <br>
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1.  Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes). <br>
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10.  Add 900µl ice cold M17+ and keep on ice for 10 mins. <br>
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2.  Add 900µl ice cold M17+ and keep on ice for 10 mins. <br>
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::*(0.5M(.17g)Sucrose + 0.5%(15µl)Glucose+20mM(10µl)MgCl+0.2mM(10µl)CaCl). <br>
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:*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>
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11.  Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br>
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3.  Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br>
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12Add entire subculture to GM17 with antibiotic. <br>
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4Plate with appropriate antibiotic. <br>
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::*1ug/ml Erm for plates. <br>
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::*5ug/ml Erm for culture.
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[[Category:Protocol]]
[[Category:Protocol]]

Current revision

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).

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.

3. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours.
4. Plate with appropriate antibiotic.

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