Team:TU-Delft/Protocol 5

From 2013.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 2: Line 2:
<html>
<html>
-
 
+
<a name="protocol_5"></a>
<br>
<br>
<p align="justify">
<p align="justify">
Line 8: Line 8:
<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
<ol>
<ol>
-
<li> Plasmid DNA</li>
+
 
-
<li> Restriction Enzymes</li>
+
    <li>plasmid DNA or PCR product </li>
-
<li> 1.5 mL Tubes</li>
+
    <li>restriction enzymes (Roche and BioLabs) </li>
-
<li>     NE Buffer 2</li>
+
    <li>buffer (10x) </li>
-
<li>     BSA</li>
+
    <li>H2O </li>
-
<li>     Distilled water</li>
+
    <li>water bath at 37 °C </li>
 +
    <li>heat block or water bath at 65 °C  </li>
 +
 
</ol>
</ol>
<br>
<br>
-
<h4 align="left">Prodecure:</h4>
+
<h4 align="left">Procedure:</h4>
<ol>
<ol>
-
<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>
+
 
-
<li> Add 5 μL NE Buffer 2 and 0.5 μL  of BSA. </li>
+
<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> There should be a total volume of 50 μL. Mix well and spin down briefly.</li>
+
 
-
<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>
+
<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>
 +
</div>
 +
 
</ol>
</ol>
<br>
<br>
-
Run a portion of the digest on a gel (8ul, 100ng)to check that both plasmid backbone and part length are accurate.
+
<ol>Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.</ol>
-
The next step would be to carry out ligation of the digested pieces of DNA.
+
<br>
 +
<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>
<br>
<br>
</p>
</p>
-
 
</html>
</html>

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.

Retrieved from "http://2013.igem.org/Team:TU-Delft/Protocol_5"