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TOP10 chemically competent cells: Difference between revisions
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TOP10 chemically competent cells (view source)
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===Preparing competent cells=== | ===Preparing competent cells=== | ||
* Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at | * Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 | ||
** This takes approximately 16 hours. | ** This takes approximately 16 hours. | ||
** Controlling the temperature makes this a more reproducible process, but is not essential. | ** Controlling the temperature makes this a more reproducible process, but is not essential. | ||
** Room temperature will work. You can adjust this temperature somewhat to fit your schedule | ** Room temperature will work. You can adjust this temperature somewhat to fit your schedule | ||
** Aim for lower, not higher OD if you can't hit this mark | ** Aim for lower, not higher OD if you can't hit this mark | ||
* Centrifuge at | * Centrifuge at 3000rpm at 4°C for 10 minutes in a flat bottom centrifuge bottle. | ||
** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend | ** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend | ||
** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer | ** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer | ||
* Discard supernatant by pouring out slowly and pipeting | * Discard supernatant by pouring out slowly and pipeting remaining supernatent | ||
* Gently resuspend in 80 ml of ice cold CCMB80 buffer | * Gently resuspend in 80 ml of ice cold CCMB80 buffer | ||
** sometimes this is less than completely gentle. It still works. | ** sometimes this is less than completely gentle. It still works. | ||
