Team:SDU-Denmark/Protocols/Competent-cells

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=Protocol for making competent cells (E.coli)=
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=Protocol for making cells (E.coli) competent for transformation=
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(Cells are kept on ice at all times!! If the cells temperature rises above ~5º C they'll lose their competency!)
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# 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
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# Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
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# Cool cells on ice.
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# Harvest the cells in screwcap tubes (4 × 10 ml).
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# Pour away the supernatant and keep the pellet on ice.
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# Wash the cells with 10 ml cold 50mM CaCl2.
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# The cells are being distributed in eppendorf tubes of 200 µl.
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# Add 41.7 µl 87% glycerol and mix well.
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# Store at -80º C.
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=Protocol for making cells (E. coli) competent for electroporation=
   
   
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  # 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
+
(Cells are kept on ice at all times!! If the cells temperature rises above ~5º C they'll lose their competency!)
-
  # Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
+
 
-
  # Cool cells on ice.
+
#      2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
-
  # Harvest the cells in screwcap tubes (4 × 10 ml).
+
#     Incubate at 37˚C on shaker until OD450=0.5-0.7
-
  # Pour away the supernatant and keep the pellet on ice.
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#     Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
-
  # Wash the cells with 10 ml cold 50mM CaCl2.
+
#     Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
-
  # The cells are being distributed in eppendorf tubes of 200 µl.
+
#     Resuspend in 100ml ice cold dH2O. Harvest as in 3.
-
  # Add 41.7 µl 87% glycerol and mix well.
+
#     Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
-
  # Store at -80 º C.
+
#     Resuspend in ice cold 10% glycerol until a total volume of 1ml. Cell concentration should be 1-3x1010 cells/ml.
 +
#     Cells are distributed in tubes with 40ul in each and kept at -80˚C.

Latest revision as of 10:05, 17 August 2009

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Protocol for making cells (E.coli) competent for transformation

(Cells are kept on ice at all times!! If the cells temperature rises above ~5º C they'll lose their competency!)

  1. 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
  2. Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
  3. Cool cells on ice.
  4. Harvest the cells in screwcap tubes (4 × 10 ml).
  5. Pour away the supernatant and keep the pellet on ice.
  6. Wash the cells with 10 ml cold 50mM CaCl2.
  7. The cells are being distributed in eppendorf tubes of 200 µl.
  8. Add 41.7 µl 87% glycerol and mix well.
  9. Store at -80º C.

Protocol for making cells (E. coli) competent for electroporation

(Cells are kept on ice at all times!! If the cells temperature rises above ~5º C they'll lose their competency!)

  1. 2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
  2. Incubate at 37˚C on shaker until OD450=0.5-0.7
  3. Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
  4. Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
  5. Resuspend in 100ml ice cold dH2O. Harvest as in 3.
  6. Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
  7. Resuspend in ice cold 10% glycerol until a total volume of 1ml. Cell concentration should be 1-3x1010 cells/ml.
  8. Cells are distributed in tubes with 40ul in each and kept at -80˚C.
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