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Team:Grinnell/Notebook/Protocols
From 2011.igem.org
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<html><h3><a name="Competent"></a>Competent Cells</h3> | <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 (~ | + | <li>Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10^8 cells/mL, OD600 = 0.3-0.5).</li> |
<li>Chill cells on ice for 15-120min.</li> | <li>Chill cells on ice for 15-120min.</li> | ||
<li>Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.</li> | <li>Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.</li> | ||
Revision as of 00:19, 9 June 2011
Competent Cells
- Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10^8 cells/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° 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° 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° C for 90sec.
- Incubate tubes on ice for 2min.
- Add 300mL LB to cells and incubate shaking at 37° C for 1hr.
- Spread cells on selective media
- Incubate plates overnight at 37° C.
