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TOP10 chemically competent cells: Difference between revisions
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→Preparing competent cells
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* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/l) | * 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/l) | ||
* 10% glycerol (100 ml/l) | * 10% glycerol (100 ml/l) | ||
* adjust pH to 6.4 with 0.1N HCl | * adjust pH DOWN to 6.4 with 0.1N HCl if necessary | ||
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. | ** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. | ||
* sterile filter and store at 4C. | * sterile filter and store at 4C. | ||
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** This is at 10 pg/μl or 10<sup>-5</sup> μg | ** This is at 10 pg/μl or 10<sup>-5</sup> μg | ||
* Hold on ice 0.5 hours | * Hold on ice 0.5 hours | ||
* Heat shock 60 sec | * Heat shock 60 sec at 42C | ||
* Add 250 μl SOC | * Add 250 μl SOC | ||
* Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated | * Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated | ||
** using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. | |||
* Plate 20 μl on AMP plates using 3.5 mm glass beads | * Plate 20 μl on AMP plates using 3.5 mm glass beads | ||
* Transformation efficiency is 15 x colony count x 10<sup>5</sup> | * Transformation efficiency is 15 x colony count x 10<sup>5</sup> | ||
