Team:TU-Delft/Protocol 5

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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_5"></a>
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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">Restriction digestion</h2>
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</p>
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<h4 align="left">Requirements:</h4>
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<ol>
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<ul style="list-style-type: circle">
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    <li>plasmid DNA or PCR product </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>restriction enzymes (Roche and BioLabs) </li>
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    <li>buffer (10x) </li>
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    <li>H2O </li>
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    <li>water bath at 37 °C </li>
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    <li>heat block or water bath at 65 °C  </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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</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> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_3" style="text-decoration: none""><font
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<li> Digestions (cutting 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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color="#0080FF" size="3"> Making glycerol stocks</font></a> </li>
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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_4" style="text-decoration: none""><font
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<table border="1" bordercolor="#CC3300" style="background-color:white" width="70%" cellpadding="3" cellspacing="0">
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color="#0080FF" size="3"> Miniprep Protocol</font></a> </li>
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<tr>
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                        <td> Component </td>
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                        <td> Sample</td>
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                    </tr>
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<tr>
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                        <td> DNA </td>
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                        <td> x μL (up to 1,0 μg) </td>
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                    </tr>
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<tr>
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                        <td> Buffer (10x)  </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> Restriction enzymes </td>
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                        <td> x μL (10 units/μg DNA = 1 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> 20-25 μL  </td>
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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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<br>
 
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<p align="justify">
 
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<h2 align="center">Restriction digestion</h2>
 
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<h4 align="left">Requirements:</h4>
 
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<ol>
 
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<li> Plasmid DNA</li>
 
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<li> Restriction Enzymes</li>
 
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<li> 1.5 mL Tubes</li>
 
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<li>    NE Buffer 2</li>
 
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<li>    BSA</li>
 
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<li>      Distilled water</li>
 
</ol>
</ol>
<br>
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<h4 align="left">Prodecure:</h4>
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<ol>Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.</ol>
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<ol>
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<li> Add 25 μL of plasmid DNA and suitable amount of Distilled water to make up total volume to 50 μL. The amount of plasmid DNA to take depends upon the concentration of the DNA solution. </li>
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<li> Add 5 μL NE Buffer 2 and 0.5 μL  of BSA. </li>
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<li> There should be a total volume of 50 μL. Mix well and spin down briefly.</li>
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<li> Incubate the restriction digest at 37°C for 30 min, and then 80°C for 20 min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid. </li>
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</ol>
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<br>
<br>
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Run a portion of the digest on a gel (8ul, 100ng)to check that both plasmid backbone and part length are accurate.
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<ol> Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly. </ol>
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The next step would be to carry out ligation of the digested pieces of DNA.
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Latest revision as of 15:27, 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.



Restriction digestion

Requirements:

  1. plasmid DNA or PCR product
  2. restriction enzymes (Roche and BioLabs)
  3. buffer (10x)
  4. H2O
  5. water bath at 37 °C
  6. heat block or water bath at 65 °C

Procedure:

  1. Digestions (cutting 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 x μL (up to 1,0 μg)
    Buffer (10x) x μL (for 1×)
    Restriction enzymes x μL (10 units/μg DNA = 1 µL)
    H2O x μL
    20-25 μL

    Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.

    Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.

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