Team:TU-Delft/Protocol 1

From 2013.igem.org

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<h2 align="center">Transforming Parts from Distribution kit</h2>
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<h4 align="left">Requirements</h4>
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<li>    competent cells </li>
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<li>      SOC medium (warmed to room temperature) </li>
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<li>      Plasmid DNA or DNA ligation mix </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>      water bath at 42 °C </li> 
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<li>      shaking incubator at 37 °C.  </li>
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<h4>Procedure: </h4>
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<h4 align="left">Procedure</h4>
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<li> Grow the cells containing the construct    <a href="http://parts.igem.org/Part:BBa_K1022100" style="text-decoration: none"" target="_blank">BBa_K1022100</a> (pBAD AIP Receiver GFP, fig. 2) overnight. </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> Make 1/50 dilutions of the overnight grown culture. Check for OD at 600nm till 0.1.</li>  
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<li> Add 10 μL of milliQ. </li>
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<li> Then induce with 0.1% Arabinose. Keep aside samples with No Arabinose, No AIPs induction to use as control. BL21 cells and Const GFP are also used as negative and positive controls.</li>
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<li> Pipette up and down several times, let sit for a few minutes. </li>
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<li> Check the OD till 0.5 and then induce with AIP to a final concentration of 1 µM and 10 µM. </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> After 3 hours of incubation time, check for GFP signals on the FACS (Fluorescence-activated cell sorting). </li>
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<li> Incubate tube vial on ice for 30 minutes</li>
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<li> Heat shock the transformation the water bath at 42⁰C for 30 sec.</li>
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<li> Incubate on ice for 2 min. </li>
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<li> Add 220 µL SOC Media to the transformation.</li>
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<li> Incubate the transformation at 37⁰C for 1 hour. </li>
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<li> Plate out 20 µL of transformation with 10% and 90% on separate agar plates with appropriate antibiotics.</li>
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<li> Incubate the agar plate overnight (14-16 hours) at 37⁰C.</li>
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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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