Lactococcus transformation

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Preparing Electrocompetent Cells
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===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.
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3.  Grow for ~4 hours until OD600 ~ 0.7. <br>
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4.  Chill culture on ice for 10 mins.
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4.  Chill culture on ice for 10 mins. <br>
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4.  Centrifuge cells for 15 mins at 3000g
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4.  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)
+
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.
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8.  Separate into 100µl aliquots and store at -80°C until use. <br>
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Electro-Transformation
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===Electro-Transformation===
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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>
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11.  Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours.  
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11.  Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours. <br>
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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).

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.
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