Team:TU-Delft/Protocol 5

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<h2 align="center">Restriction digestion</h2>
<h4 align="left">Requirements:</h4>
<ol>

    <li>plasmid DNA or PCR product </li>
    <li>restriction enzymes (Roche and BioLabs) </li>
    <li>buffer (10x) </li>
    <li>H2O </li>
    <li>water bath at 37 °C </li>
    <li>heat block or water bath at 65 °C  </li>

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<h4 align="left">Procedure:</h4>
<ol>

<li> Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.</li>

<li>Reaction for one sample: </li>

<table border="1" bordercolor="#CC3300" style="background-color:white" width="70%" cellpadding="3" cellspacing="0">
	<tr>
                        <td> Component </td>
                        <td> Sample</td>
                    </tr>
 <tr>
                        <td> DNA </td>
                        <td> x μL (up to 1,0 μg) </td>
                    </tr>
 <tr>
                        <td> Buffer (10x)  </td>
                        <td> x μL (for 1×)  </td>
                    </tr>
 <tr>
                        <td> Restriction enzymes </td>
                        <td> x μL (10 units/μg DNA = 1 µL) </td>
                    </tr>
 <tr>
                        <td> H2O  </td>
                        <td> x μL  </td>
                    </tr>
 <tr>
                        <td>  </td>
                        <td> 20-25 μL   </td>
                       </tr>

</table>
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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> 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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