Lactococcus transformation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Preparing Electrocompetent Cells)
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 (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>
-
::*(25ml M17 + 350µl glucose solution +5g Sucrose + 0.5g glycine). <br>
+
::*(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>
Line 13: Line 13:
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===

Revision as of 17:57, 5 April 2011

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.

  • (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

12. Add 5µl of DNA and electroporate at 1200 volts (1mm Cuvettes).
13. 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).

14. Subculture 100µl into 900ul prewarmed M17+ and incubate for 2 hours.
15. Add entire subculture to GM17 with antibiotic.

  • 1ug/ml Erm for plates.
  • 5ug/ml Erm for culture.
Personal tools