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TOP10 chemically competent cells: Difference between revisions
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TOP10 chemically competent cells (view source)
Revision as of 22:56, 8 November 2006
, 8 November 2006→Preparing competent cells
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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 20 C to an OD of 0.3 | * Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 20 C to an OD 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. | |||
** 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 | |||
* Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle. | * Centrifuge at 3000g / 4C 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 | ||
* 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. | |||
* Incubate on ice 20 minutes | * Incubate on ice 20 minutes | ||
* Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer. | * Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer. | ||
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** Flash freezing does not appear to be necessary | ** Flash freezing does not appear to be necessary | ||
* Test competence (see below) | * Test competence (see below) | ||
** Good cells should yield around 100 colonies | ** Good cells should yield around 100 - 400 colonies | ||
===CCMB80 buffer=== | ===CCMB80 buffer=== | ||
