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Checking Transformation Efficiency of Chemically Competent Cells
Adapted from Molecular Cloning: A Laboratory Manual 3rd Ed., Sambrook and Russell (2001) |
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- Shaking Incubator or Shaking Platform and 1L flask clamps
- 42°C Heating Bath or Block
- 37°C Incubator
- Colony Counter
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- Agar plates with appropriate antibiotic
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- Competent cells prepared using the Inoue method or another method
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- 10pg / μL solution of a standard plasmid (e.g. pUC19)
- SOC Medium
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- ) Transform 10pg of a standard plasmid (e.g. pUC19) in 50μL of cells using the standard transformation protocol.
- ) Plate 50uL of transformed cells in triplicate on appropriate antibiotic
- ) Grow plates overnight, and count colonies the next morning
- ) Average colony counts for the three plates; efficiency = (# colonies) x 106 cfu / μg
- ) Expected efficiency for the Inoue protocol is 1-3 x 108 colonies / μg of plasmid
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- Transform 10 pg of a standard plasmid (e.g. pUC19) in 50 µL of cells using the standard transformation protocol.
- Plate 50 µL of transformed cells in triplicate on appropriate antibiotic
- Grow plates overnight, and count colonies the next morning
- Average colony counts for the three plates; efficiency = (average # colonies) x 106 cfu / μg
- Expected efficiency for chemically competent cells prepared by the Inoue protocol is 1-3 x 108 colonies / μg of plasmid
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< | -- Main.KateElston - 25 Oct 2018 |
