Team:TU-Delft/Protocol 6

From 2013.igem.org

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<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
<ol>
<ol>
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<li> Cut Insert DNA</li>
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    <li> digested plasmid DNA or PCR product </li>
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<li>     Cut Vector DNA</li>
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    <li> T4 ligation buffer (10x) (Fermentas) </li>
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<li> Ligase buffer</li>
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    <li>T4 ligase (Fermentas) </li>
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<li> T4 DNA ligase</li>
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    <li>H2O </li>
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<li>     Distilled water</li>
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    <li>water bath at 16 °C  </li>
</ol>
</ol>
<br>
<br>
<h4 align="left">Prodecure:</h4>
<h4 align="left">Prodecure:</h4>
<ol>
<ol>
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<li> Calculate the amount of vector DNA and insert DNA to add based on the ratio of the DNA and vector(3:1),if insert DNA size is 5 times smaller than vector DNA or 1:1 ratio if both sizes are same. </li>
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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>
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<li> Add suitable amount of distilled water to make up total volume to 50 μL. </li>
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<li> Add 1 μL T4 DNA ligase buffer. </li>
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<li> Reaction for one sample:  </li>
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<li> Add 1 μL T4 DNA ligase. </li>
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<li> Ligate at 16°C overnight or leave it at room temperature for 4 hours.  </li>  
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<table border="1" bordercolor="#CC3300" style="background-color:white" width="70%" cellpadding="3" cellspacing="0">
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<tr>
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<td><b>Component</b></td>
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<td><b>Sample</b></td>
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 +
</tr>
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<tr>
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<td> DNA insert </td>
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<td> x μL </td>
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</tr>
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        <tr>
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<td> DNA vector  </td>
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<td> x μL </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> T4 Ligation buffer (10×)  </td>
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<td> x μL (for 1×)  </td>
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 +
</tr>
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        <tr>
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<td> T4 Ligase  </td>
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<td> 1.0 μL  </td>
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 +
</tr>
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        <tr>
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<td> H2O </td>
 +
<td> x μL </td>
 +
 +
</tr>
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        <tr>
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<td> </td>
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<td> 10-15 μL  </td>
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 +
</tr>
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</table>
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</ol>
</ol>
<br>
<br>

Revision as of 15:15, 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

Prodecure:

  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 next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.

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