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Team:SDU-Denmark/Protocols/Competent-cells
From 2009.igem.org
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(→Protocol for making competent cells (E.coli)) |
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=Protocol for making competent cells (E.coli)= | =Protocol for making competent cells (E.coli)= | ||
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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!) | ||
# 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium. | # 600 µl overnight (ON) Top10 E. coli culture is added to 60 ml Luria-Bertani (LB) medium. | ||
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# The cells are being distributed in eppendorf tubes of 200 µl. | # The cells are being distributed in eppendorf tubes of 200 µl. | ||
# Add 41.7 µl 87% glycerol and mix well. | # Add 41.7 µl 87% glycerol and mix well. | ||
| - | # Store at - | + | # Store at -80º C. |
Revision as of 15:20, 24 July 2009
Protocol for making competent cells (E.coli)
(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.
