Team:TU-Delft/Protocol 6

From 2013.igem.org

(Difference between revisions)
 
(5 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:TU-Delft/Templates/Navigation}}
+
{{:Team:TU-Delft/ProtocolList}}
-
{{:Team:TU-Delft/Templates/Style}}
+
-
{{:Team:TU-Delft/Templates/Frog}}
+
-
{{:Team:TU-Delft/Templates/Logo}}
+
-
 
+
-
 
+
-
 
+
-
 
+
-
<div style="margin-left:30px;margin-right:30px; width:900px;float:left;"> 
+
-
<h2 align="center">Protocols</h2>
+
<html>
<html>
-
 
+
<a name="protocol_6"></a>
-
 
+
-
<div style="margin-left:50px;margin-right:50px;float:left;display:inline-block;"> 
+
-
 
+
-
<p align="justify">
+
-
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.
+
-
</p>
+
-
 
+
-
<ul style="list-style-type: circle">
+
-
  <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>
+
-
 
+
-
<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>
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_3" style="text-decoration: none""><font
+
-
color="#0080FF" size="3"> Making glycerol stocks</font></a> </li>
+
-
 
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_4" style="text-decoration: none""><font
+
-
color="#0080FF" size="3"> Miniprep Protocol</font></a> </li>
+
-
 
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_5" style="text-decoration: none""><font
+
-
color="#0080FF" size="3"> Restriction digestion</font></a> </li>
+
-
 
+
-
 
+
-
 
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_6" style="text-decoration: none""><font
+
-
color="#0080FF" size="3"> Ligation</font></a> </li>
+
-
 
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_7" style="text-decoration: none"" ><font
+
-
color="#0080FF" size="3"> Gel Extraction Procedure</font></a> </li>
+
-
 
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_8" style="text-decoration: none""><font
+
-
color="#0080FF" size="3">  PCR Purification</font></a> </li>
+
-
 
+
-
<li> <a href="https://2013.igem.org/Team:TU-Delft/Protocol_9" style="text-decoration: none""><font
+
-
color="#0080FF" size="3">  Tricine Gels</font></a> </li>
+
-
</ul>
+
-
 
+
-
 
+
<br>
<br>
Line 60: Line 9:
<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
<ol>
<ol>
-
<li> Cut Insert DNA</li>
+
    <li> digested plasmid DNA or PCR product </li>
-
<li>     Cut Vector DNA</li>
+
    <li> T4 ligation buffer (10x) (Fermentas) </li>
-
<li> Ligase buffer</li>
+
    <li>T4 ligase (Fermentas) </li>
-
<li> T4 DNA ligase</li>
+
    <li>H2O </li>
-
<li>     Distilled water</li>
+
    <li>water bath at 16 °C  </li>
</ol>
</ol>
<br>
<br>
-
<h4 align="left">Prodecure:</h4>
+
<h4 align="left">Procedure:</h4>
<ol>
<ol>
-
<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>
+
<li> Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. </li>
-
<li> Add suitable amount of distilled water to make up total volume to 50 μL. </li>
+
 
-
<li> Add 1 μL T4 DNA ligase buffer. </li>
+
<li> Reaction for one sample:  </li>
-
<li> Add 1 μL T4 DNA ligase. </li>
+
 
-
<li> Ligate at 16°C overnight or leave it at room temperature for 4 hours.  </li>  
+
<table border="1" bordercolor="#CC3300" style="background-color:white" width="70%" cellpadding="3" cellspacing="0">
 +
<tr>
 +
<td><b>Component</b></td>
 +
<td><b>Sample</b></td>
 +
 +
</tr>
 +
<tr>
 +
<td> DNA insert </td>
 +
<td> x μL </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> DNA vector  </td>
 +
<td> x μL </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> T4 Ligation buffer (10×)  </td>
 +
<td> x μL (for 1×)  </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> T4 Ligase  </td>
 +
<td> 1.0 μL  </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> H2O </td>
 +
<td> x μL </td>
 +
 +
</tr>
 +
        <tr>
 +
<td> </td>
 +
<td> 10-15 μL  </td>
 +
 +
</tr>
 +
</table>
 +
 
</ol>
</ol>
<br>
<br>
-
The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.
+
<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.
 +
 
 +
Transform circa half of the ligation mix. Incubate at 16 °C o/n. </ol>
<br>
<br>
</p>
</p>
</html>
</html>

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.

Retrieved from "http://2013.igem.org/Team:TU-Delft/Protocol_6"