Team:INSA-Lyon/Protocols/Ligation

From 2010.igem.org

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<span style="font-style : italic">If it follows a digestion without purification: <u>thermoinactivation 20 min at 70°C </u></span>
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<span style="font-style : italic">If it follows a digestion without purification: <u>20 minutes of thermoinactivation at 70°C </u></span>
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Revision as of 17:24, 20 October 2010







Protocols


Currently under construction



  1. Competent cells
  2. Transformation
  3. DNA extraction
  4. Digestion
  5. Ligation
  6. Measure of temperature and shaking speed influence
  7. Measure of osmotic pressure influence
  8. Granules extraction and intein cleavage
  9. Biofilms quantification
  10. Extra






    Ligation



      If it follows a digestion without purification: 20 minutes of thermoinactivation at 70°C

    • Add x µL DNA (the entire digestion gel purification for part ligation) Plasmid: 50 to 200 ng; Insert: 100 ng (0,5 kb) to 1 µg (10 kb))
    • Add 2 µL Buffer T4 DNA ligase (Attention: this buffer contains ATP, defrost on ice)
    • Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last
    • Add sterile water qs 20 µL

      • Incubate 3h at room temperature or overnight at 15°C

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