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Team:Grinnell/Notebook/Protocols
From 2011.igem.org
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| - | <html><h3><a name="Competent"> | + | <html><h3><a name="Competent"></a>Competent Cells</h3> |
<ol> | <ol> | ||
<li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li> | <li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).</li> | ||
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<li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.</li> | <li>Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.</li> | ||
</ol> | </ol> | ||
| - | <h3><a name="Transformation"> | + | <h3><a name="Transformation"></a>Plasmid Transformation</h3> |
<ol> | <ol> | ||
<li>Thaw 100uL aliquots of competent cells on ice.</li> | <li>Thaw 100uL aliquots of competent cells on ice.</li> | ||
Revision as of 21:50, 8 June 2011
Competent Cells
- Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5x10^8cells/mL, OD600 = 0.3-0.5).
- Chill cells on ice for 15-120min.
- Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4 degrees C. Discard supernatant.
- Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
- Harvest cells by cetrifugation. Discard supernatant.
- Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80 degrees C.
Plasmid Transformation
- Thaw 100uL aliquots of competent cells on ice.
- Add 10uL DNA to cells.
- Incubate tubes on ice for 30min.
- Incubate tubes at 42 degrees C for 90sec.
- Incubate tubes on ice for 2min.
- Add 300mL LB to cells and incubate shaking at 37 degrees C for 1hr.
- Spread cells on selective media
- Incubate plates overnight at 37 degrees C.
