Team:TU-Delft/Protocol 6

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<div style="margin-left:30px;margin-right:30px; width:900px;float:left;"> 
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<h2 align="center">Protocols</h2>
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<a name="protocol_6"></a>
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<div style="margin-left:50px;margin-right:50px;float:left;display:inline-block;"> 
 
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Our project deals with <i>E.coli</i> cells which sense Auto-inducing peptides (AIPs) from the <i>Staphylococcus aureus</i> and starts producing Antimicrobial peptides in order to kill the <i>Staphylococcus aureus</i>. Different protocols used during the project are described below.
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<h2 align="center">Ligation</h2>
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</p>
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<h4 align="left">Requirements:</h4>
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<ol>
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    <li> digested plasmid DNA or PCR product </li>
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    <li> T4 ligation buffer (10x) (Fermentas) </li>
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    <li>T4 ligase (Fermentas) </li>
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    <li>H2O </li>
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    <li>water bath at 16 °C  </li>
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</ol>
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<br>
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<h4 align="left">Procedure:</h4>
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<ol>
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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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<ul style="list-style-type: circle">
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<li> Reaction for one sample:  </li>
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  <li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_1" style="text-decoration: none""><font color="#0080FF" size="3">Transforming Parts from Distribution kit</font></a> </li>
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  <li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol2" style="text-decoration: none""><font color="#0080FF" size="3">Growing the Single Colonies from the Agar Plates</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_3" style="text-decoration: none""><font
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color="#0080FF" size="3"> Making glycerol stocks</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_4" style="text-decoration: none""><font
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color="#0080FF" size="3"> Miniprep Protocol</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_5" style="text-decoration: none""><font
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color="#0080FF" size="3"> Restriction digestion</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_6" style="text-decoration: none""><font
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color="#0080FF" size="3"> Ligation</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_7" style="text-decoration: none"" ><font
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color="#0080FF" size="3"> Gel Extraction Procedure</font></a> </li>
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<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_8" style="text-decoration: none""><font
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color="#0080FF" size="3">  PCR Purification</font></a> </li>
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</ul>
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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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<td> DNA insert </td>
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<td> x μL  </td>
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        <tr>
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<td> DNA vector  </td>
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<td> x μL  </td>
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        <tr>
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<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>
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<td> x μL  </td>
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        <tr>
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<td> </td>
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<td> 10-15 μL  </td>
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</ol>
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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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<h2 align="left">Ligation</h2>
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<h4 align="left">Requirements:</h4>
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1. Cut Insert DNA
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2.      Cut Vector DNA
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3. Ligase buffer
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4. T4 DNA ligase
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5.      Distilled water
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<br>
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<h4 align="left">Prodecure:</h4>
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1. 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. <br>
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2. Add suitable amount of distilled water to make up total volume to 50 μL. <br>
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3. Add 1 μL T4 DNA ligase buffer. <br>
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4. Add 1 μL T4 DNA ligase. <br>
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5. Ligate at 16°C overnight or leave it at room temperature for 4 hours. <br>  
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<br>
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The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.
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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.

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