Difference between revisions of "Team:Bielefeld-CeBiTec/Protocols"

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                    <a data-toggle="collapse" href="#CFPSprotocols">CFPS protocols</a>
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<ul><li> Cell harvest </li>
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        <li> The following harvest protocol mainly orientates to the procedures in <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013">Sun et al. 2013</a> and <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#KwonJewett2015">Kwon and Jewett 2015</a></li>
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        <li> Gather all materials needed: Harvest tubes and falcon tubes, washing buffer, DTT aliquot, liquid nitrogen, and a lot of ice. Start the harvest when <i>E. coli</i> culture reaches mid- to late exponential growth phase. For the <i>E. coli</i> we used in our experiments and cultivations, the mid- to late exponential growth phase was reached at an OD<sub>600</sub> of 3-4 (see growth curves in <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Notebook/CFPS">protocol section</a>).</li>
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        <li> harvest protocol - <b>keep everthing on ice between the steps!</b></li>
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                <li> Transfer culture into prechilled and weighted harvest tubes or falcons</li>
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                <li> Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM</li>
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                <li> Discard supernatant and weight pellets </li>
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                <li> Add about 10 mL of S30 washing buffer and resuspend cells by vortexing and vigourous shaking. For us, cycles of 15 s vortexing/shaking and 30 s resting on ice worked well.  </li>
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                <li> Centrifugate: 5000x g, 4 °C, 12 min</li>
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                <li> Discard supernatant</li>
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                <li> Repeat steps 4 to 6 two times </li>
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                <li> Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.</li>
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                <li> Flash freeze pellets in liquid nitrogen and store at -80 °C, unless you want to proceed with sonification directly. </li>
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Revision as of 09:49, 15 August 2015

iGEM Bielefeld 2015


Protocols



CFPS protocols

  • Cell harvest
    • The following harvest protocol mainly orientates to the procedures in Sun et al. 2013 and Kwon and Jewett 2015
    • Gather all materials needed: Harvest tubes and falcon tubes, washing buffer, DTT aliquot, liquid nitrogen, and a lot of ice. Start the harvest when E. coli culture reaches mid- to late exponential growth phase. For the E. coli we used in our experiments and cultivations, the mid- to late exponential growth phase was reached at an OD600 of 3-4 (see growth curves in protocol section).
    • harvest protocol - keep everthing on ice between the steps!
      1. Transfer culture into prechilled and weighted harvest tubes or falcons
      2. Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM
      3. Discard supernatant and weight pellets
      4. Add about 10 mL of S30 washing buffer and resuspend cells by vortexing and vigourous shaking. For us, cycles of 15 s vortexing/shaking and 30 s resting on ice worked well.
      5. Centrifugate: 5000x g, 4 °C, 12 min
      6. Discard supernatant
      7. Repeat steps 4 to 6 two times
      8. Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.
      9. Flash freeze pellets in liquid nitrogen and store at -80 °C, unless you want to proceed with sonification directly.

Colony PCR

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DNA Purification

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Gibson Assembly

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PCR

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Plasmid Isolation

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Standard BioBrick Assembly

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Transformation via Electroporation

  • Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
  • Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
  • Store cells on ice for 1 minute
  • Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
  • Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
  • Plate on selective LB-Medium
  • Incubate over night at 37 °C

Transformation via Heat Shock

  • Thaw 100 µl chemo competent E. coli cells on ice
  • Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
  • Store cells on ice for 10-30 min on ice
  • Heat shock for 90 seconds at 42 °C
  • Store reaction on ice for 60 seconds
  • Optional: Preheat SOC medium to 37 °C
  • Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
  • Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
  • Incubate at 37 °C over night

2015 InterLab Protocol

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