Team:UEA-JIC Norwich/Methods

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<h2>High Efficiency Transformation Protocol</h2>
<h2>High Efficiency Transformation Protocol</h2>
 +
Perform steps 1-13:
Perform steps 1-13:
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1. Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes.
+
<ol>
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2. Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right).
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<li>Thaw a tube of NEB 5-alpha Competent E.coli cells on ice for 10 minutes.</li>
-
3. Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.
+
<li>Mark the location of the Biobrick well (letters from top to bottom, numbers from left to right).</li>
-
4. Inoculate with DNA ( 1 μl) to the competent cells.
+
<li>Resuspend specific biobrick part (1 μl) with distilled water (20 μl). Aspirate up and down a few times.</li>
-
5. Place the mixture on ice for 30 minutes. Do not mix.
+
<li>Inoculate with DNA ( 1 μl) to the competent cells.</li>
-
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
+
<li>Place the mixture on ice for 30 minutes. Do not mix.</li>
-
7. Place on ice for 5 minutes. Do not mix
+
<li>Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.</li>
-
8. Pipette 950 μl of room temperature SOC into the mixture.
+
<li>Place on ice for 5 minutes. Do not mix.</li>
-
9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
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<li>Pipette 950 μl of room temperature SOC into the mixture.</li>
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10. Warm selection plates to 37°C.
+
<li>Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.</li>
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11. Do serial dilutions (2-3) at 105 . Mix cells, flick/invert gently.
+
<li>Warm selection plates to 37°C.</li>
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12. Spread (100 μl) on agar plates of each dilution.
+
<li>Do serial dilutions (2-3) at 105 . Mix cells, flick/invert gently.</li>
-
13. Incubate overnight at 37°C.
+
<li>Spread (100 μl) on agar plates of each dilution.</li>
 +
<li>Incubate overnight at 37°C.</li>

Revision as of 15:06, 20 June 2011

High Efficiency Transformation Protocol

Perform steps 1-13:
  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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