Difference between revisions of "Team:Tuebingen/Experiments"

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<h2> Protocols</h2>
 
<h2> Protocols</h2>
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         <h4> Prep </h4>
 
         <h4> Prep </h4>
 
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         <h4> Colony-PCR </h4>
 
         <h4> Colony-PCR </h4>
 
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             <li> Centrifuge 200&mu;l o/n culture, discard the supernatant and put the pellets in PCR tubes. </li>
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            <li> Add 5&mu;l Q5 High-Fidelity 2X MasterMix from NEB.</li>
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            <li> Add 1,8&mu; of each primer: fw/rv</li>
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            <li> Add x &mu;l water up to 10&mu;l per mixture.</li>
 
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         <h4> 3A-Assembly </h4>
 
         <h4> 3A-Assembly </h4>

Revision as of 09:34, 25 August 2015

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Protocols

Prep

Colony-PCR

  • Centrifuge 200μl o/n culture, discard the supernatant and put the pellets in PCR tubes.
  • Add 5μl Q5 High-Fidelity 2X MasterMix from NEB.
  • Add 1,8μ of each primer: fw/rv
  • Add x μl water up to 10μl per mixture.

3A-Assembly

Restriction

Transformation

  • 0,2μl EcoRI-HF
  • 0,2μl PstI
  • 1μl Buffer
  • 1μg DNA[?]
  • 4μl Plasmid
  • 2μl Cre
  • Fill up with water to 10μl.
  • Run a 1% agarose gel at 37°C for 1,5h.

PCR

Ligation

  • 5 μl Water
  • 2 μl Puffer NEB
  • 1 μl Ligase NEB
  • 2 μl Plasmid (pSB)
  • 10 μl Insert
  • 4 °C o/n

Testrestriktion

Gel extraction

1% Agarose gel

  • 3 μl Midori green
  • 100 ml TBE
  • 1g Agarose

Pouring agar plates

  • LB Agar-Agar
  • 200ml 1:1000 Chloramphenicol(11 plates)
  • 200ml 1:1000 Ampicillin(11 plates)

DNA Purification using Promega Wizard SV Gel and PCR Clean-Up System Kit

This is a modified protocol for the Wizard SV Gel and PCR Clean-Up System Kit from Promega

  • Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube.
  • Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-60°C until gel slice is completely dissolved.
  • Insert SV Minicolumn into Collection Tube.
  • Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute.
  • Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
  • Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
  • Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min.
  • Empty the Collection Tube and recentrifuge the column assembly for 1,5 min.
  • Leave the tubes open for 10 min (to let any rest of ethanol evaporate).
  • Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
  • Add 30μl of Nuclease-Free-Water (65°C) to the Minicolumn. Incubate at 65°C for 5 min. Centrifuge at 16,000xg for 1 min.
  • Discard Minicolumn and store DNA at 4°C or -20°C.

Experiments & Protocols

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project

Inspiration