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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<ol>
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<li>Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes.
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<p>1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes</p>
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<li>Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right).
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<p>2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)</p>
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<li>Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.</li>
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<p>3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times</p>
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<li>Inoculate with DNA ( 1 μl) to the competent cells.</li>
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<p>4. Inoculate with DNA ( 1 μl) to the competent cells</p>
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<li>Place the mixture on ice for 30 minutes. Do not mix.</li>
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<p>5. Place the mixture on ice for 30 minutes. Do not mix</p>
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<li>Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.</li>
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<p>6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix</p>
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<li>Place on ice for 5 minutes. Do not mix.</li>
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<p>7. Place on ice for 5 minutes. Do not mix</p>
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<li>Pipette 950 μl of room temperature SOC into the mixture.</li>
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<p>8. Pipette 950 μl of room temperature SOC into the mixture</p>
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<li>Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.</li>
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<p>9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate</p>
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<li>Warm selection plates to 37°C.</li>
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<p>10. Warm selection plates to 37°C</p>
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<li>Do serial dilutions (2-3) at 105 . Mix cells, flick/invert gently.</li>
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<p>11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently</p>
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<li>Spread (100 μl) on agar plates of each dilution.</li>
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<p>12. Spread (100 μl) on agar plates of each dilution</p>
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<li>Incubate overnight at 37°C.</li>
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<p>13. Incubate overnight at 37°C</p>
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</ol>
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Revision as of 08:50, 21 June 2011

High Efficiency Transformation Protocol

1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes

2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right)

3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times

4. Inoculate with DNA ( 1 μl) to the competent cells

5. Place the mixture on ice for 30 minutes. Do not mix

6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix

7. Place on ice for 5 minutes. Do not mix

8. Pipette 950 μl of room temperature SOC into the mixture

9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate

10. Warm selection plates to 37°C

11. Do serial dilutions (2-3) at 105. Mix cells, flick/invert gently

12. Spread (100 μl) on agar plates of each dilution

13. Incubate overnight at 37°C

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