Team:TU-Delft/Protocol 6

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  <li> Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. </li>
  <li> Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. </li>
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The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.
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<ol> The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix.
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Transform circa half of the ligation mix. Incubate at 16 °C o/n. </ol>
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Latest revision as of 15:28, 3 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.



Ligation

Requirements:

  1. digested plasmid DNA or PCR product
  2. T4 ligation buffer (10x) (Fermentas)
  3. T4 ligase (Fermentas)
  4. H2O
  5. water bath at 16 °C

Procedure:

  1. Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
  2. Reaction for one sample:
  3. Component Sample
    DNA insert x μL
    DNA vector x μL
    T4 Ligation buffer (10×) x μL (for 1×)
    T4 Ligase 1.0 μL
    H2O x μL
    10-15 μL

    The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix. Transform circa half of the ligation mix. Incubate at 16 °C o/n.