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Team:UEA-JIC Norwich/Methods
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<h2>High Efficiency Transformation Protocol</h2> | <h2>High Efficiency Transformation Protocol</h2> | ||
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| - | + | <li>Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes. | |
| - | + | <li>Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right). | |
<li>Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.</li> | <li>Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.</li> | ||
<li>Inoculate with DNA ( 1 μl) to the competent cells.</li> | <li>Inoculate with DNA ( 1 μl) to the competent cells.</li> | ||
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<li>Incubate overnight at 37°C.</li> | <li>Incubate overnight at 37°C.</li> | ||
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Revision as of 08:33, 21 June 2011
- Home
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High Efficiency Transformation Protocol
- Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes.
- Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right).
- Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.
- Inoculate with DNA ( 1 μl) to the competent cells.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 μl of room temperature SOC into the mixture.
- Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Do serial dilutions (2-3) at 105 . Mix cells, flick/invert gently.
- Spread (100 μl) on agar plates of each dilution.
- Incubate overnight at 37°C.
