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TOP10 chemically competent cells: Difference between revisions
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→Preparing competent cells
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* 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 | |||
* Gently resuspend in 80 ml of ice cold CCMB80 buffer | * Gently resuspend in 80 ml of ice cold CCMB80 buffer | ||
* Incubate on ice 20 minutes | * Incubate on ice 20 minutes | ||
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CCMB80 buffer | ===CCMB80 buffer=== | ||
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/l) | * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/l) | ||
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Measurement of competence | ===Measurement of competence=== | ||
* Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen) | * Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen) | ||
** This is at 10 pg/μl or 10<sup>-5</sup> μg | ** This is at 10 pg/μl or 10<sup>-5</sup> μg | ||
