Team:TU-Delft/Protocol 1

From 2013.igem.org

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<h2 align="center">Transforming Parts from Distribution kit</h2>
<h2 align="center">Transforming Parts from Distribution kit</h2>
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<h4 align="left">Requirements:</h4>
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<h4 align="left">Requirements</h4>
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<ol>
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<li> Distilled water</li>
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<li>   competent cells </li>
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<li>Pipettes</li>
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<li>     SOC medium (warmed to room temperature) </li>
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<li> Ice </li>
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<li>     Plasmid DNA or DNA ligation mix </li>
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<li> Competent cells</li>
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<li>     LB agar plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C </li>
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<li> SOC media</li>
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<li>     water bath at 42 °C </li>
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<li> Agar plates with Antibiotics</li>
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<li>     shaking incubator at 37 °C.  </li>
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<li> Timer</li>
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<li> Water bath</li>
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<h2 align="center">Prodecure</h2>
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<h4 align="left">Procedure</h4>
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<ol>
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<li>   Mark the location on distribution plate by looking at the right row and column as mentioned. Punch the plate with a pipette and add 10 µL of distilled water. </li>
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<li> Punch a hole with a pipette tip through the foil cover into the corresponding well of the desired BioBrick part.  </li>
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<li> Resuspend the Dry DNA on the distribution plate with distilled water. </li>
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<li> Add 10 μL of milliQ. </li>
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<li> Inoculate the competent cells with the desired DNA from the distribution plate as described in step 2. Add 1 µL of the resuspended DNA to the competent cells. Use 2 mL tubes and keep the cells on ice at all the times for a more efficient transformation. </li>
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<li> Pipette up and down several times, let sit for a few minutes. </li>
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<li> Incubate the transformation on ice for 5 min. </li>
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<li> Add 50-100 ng DNA into a 20 μL competent E.coli, and mix gently. Do not mix by pipetting up and down! </li>
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<li> Incubate tube vial on ice for 30 minutes</li>
<li> Heat shock the transformation the water bath at 42⁰C for 30 sec.</li>
<li> Heat shock the transformation the water bath at 42⁰C for 30 sec.</li>
<li> Incubate on ice for 2 min. </li>
<li> Incubate on ice for 2 min. </li>
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<li> Add 200 µL SOC Media to the transformation.</li>
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<li> Add 220 µL SOC Media to the transformation.</li>
<li> Incubate the transformation at 37⁰C for 1 hour. </li>
<li> Incubate the transformation at 37⁰C for 1 hour. </li>
<li> Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.</li>
<li> Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.</li>
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<br>
<br>
Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.  
Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.  
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Latest revision as of 10:52, 4 October 2013



Protocols

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.


Transforming Parts from Distribution kit

Requirements

  1. competent cells
  2. SOC medium (warmed to room temperature)
  3. Plasmid DNA or DNA ligation mix
  4. LB agar plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C
  5. water bath at 42 °C
  6. shaking incubator at 37 °C.

Procedure

  1. Punch a hole with a pipette tip through the foil cover into the corresponding well of the desired BioBrick part.
  2. Add 10 μL of milliQ.
  3. Pipette up and down several times, let sit for a few minutes.
  4. Add 50-100 ng DNA into a 20 μL competent E.coli, and mix gently. Do not mix by pipetting up and down!
  5. Incubate tube vial on ice for 30 minutes
  6. Heat shock the transformation the water bath at 42⁰C for 30 sec.
  7. Incubate on ice for 2 min.
  8. Add 220 µL SOC Media to the transformation.
  9. Incubate the transformation at 37⁰C for 1 hour.
  10. Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.
  11. Incubate the agar plate overnight (14-16 hours) at 37⁰C.

Single colonies are selected from the above plates and grown in LB media to prepare glycerol stocks and carry out Miniprep to extract the desired plasmid.

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