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TOP10 chemically competent cells: Difference between revisions
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TOP10 chemically competent cells (view source)
Revision as of 19:51, 25 August 2011
, 25 August 2011→Preparing competent cells
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===Preparing competent cells=== | ===Preparing competent cells=== | ||
* Inoculate | * Inoculate 250 ml of [[SOB]] medium with 1 ml vial of seed stock and grow at 37°C to an OD600nm of 0.5 | ||
** 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 5000rpm 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 remains | ||
* Gently resuspend in | * 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. | ||
* Incubate on ice 20 minutes | * Incubate on ice 20 minutes | ||
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* Resuspend in 10 ml of ice cold CCMB80 buffer. | * Resuspend in 10 ml of ice cold CCMB80 buffer. | ||
* Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells. | * Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells. | ||
* Add chilled CCMB80 to yield a final OD of | * Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. | ||
* Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates | * Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates | ||
* Store at -80°C indefinitely. | * Store at -80°C indefinitely. | ||
** Flash freezing does not appear to be necessary | ** Flash freezing does not appear to be necessary | ||
* Test competence (see below) | * Test competence (see below) | ||
* Thawing and | * Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. | ||
===Measurement of competence=== | ===Measurement of competence=== | ||
