Team:SDU-Denmark/Protocols/Competent-cells
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- | =Protocol for making | + | [[Team:SDU-Denmark|Home]] | [[Team:SDU-Denmark/Background|Background]] | [[Team:SDU-Denmark/Project|Project]] | [[Team:SDU-Denmark/Parts|Parts]] | [[Team:SDU-Denmark/Team|Team]] |
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+ | [[Team:SDU-Denmark/Diary|Diary]] | [[Team:SDU-Denmark/Protocols|Protocols]] | [[Team:SDU-Denmark/Downloads|Downloads]] | [[Team:SDU-Denmark/Brainstorm|Brainstorm]] | ||
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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!) | (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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# Add 41.7 µl 87% glycerol and mix well. | # Add 41.7 µl 87% glycerol and mix well. | ||
# Store at -80º C. | # Store at -80º C. | ||
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+ | =Protocol for making cells (E. coli) competent for electroporation= | ||
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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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+ | # 2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium. | ||
+ | # Incubate at 37˚C on shaker until OD450=0.5-0.7 | ||
+ | # Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C. | ||
+ | # Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3. | ||
+ | # Resuspend in 100ml ice cold dH2O. Harvest as in 3. | ||
+ | # Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3. | ||
+ | # 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!)
- 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium.
- Grows at 37º C while being shaken until the optic density (OD550) is 0,2.
- Cool cells on ice.
- Harvest the cells in screwcap tubes (4 × 10 ml).
- Pour away the supernatant and keep the pellet on ice.
- Wash the cells with 10 ml cold 50mM CaCl2.
- The cells are being distributed in eppendorf tubes of 200 µl.
- Add 41.7 µl 87% glycerol and mix well.
- 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!)
- 2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
- Incubate at 37˚C on shaker until OD450=0.5-0.7
- Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
- Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
- Resuspend in 100ml ice cold dH2O. Harvest as in 3.
- Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
- 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.