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From 2011.igem.org

Competent Cells

1. 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).
2. Chill cells on ice for 15-120min.
3. Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.
4. Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
5. Harvest cells by cetrifugation. Discard supernatant.
6. 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

1. Thaw 100uL aliquots of competent cells on ice.
2. Add 10uL DNA to cells.
3. Incubate tubes on ice for 30min.
4. Incubate tubes at 42° C for 90sec.
5. Incubate tubes on ice for 2min.
6. Add 300mL LB to cells and incubate shaking at 37° C for 1hr.
7. Spread cells on selective media
8. Incubate plates overnight at 37° C.