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TOP10 chemically competent cells: Difference between revisions
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→Measurement of competence
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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/μl | ||
** This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE | |||
* Hold on ice 0.5 hours | * Hold on ice 0.5 hours | ||
* Heat shock 60 sec at 42C | * Heat shock 60 sec at 42C | ||
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** For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies | ** For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies | ||
** Ampicillin and kanamycin appear to do fine with 1 hour growth | ** Ampicillin and kanamycin appear to do fine with 1 hour growth | ||
* Plate 20 μl on AMP plates using 3.5 mm glass beads | * Plate 20 μl on AMP plates using sterile 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> | ||
