Team:TU-Delft/Protocol 6

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<h2 align="left">Ligation</h2>
<h2 align="left">Ligation</h2>
<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
-
1. Cut Insert DNA
+
<ol>
-
2.      Cut Vector DNA
+
<li> Cut Insert DNA</li>
-
3. Ligase buffer
+
<li>    Cut Vector DNA</li>
-
4. T4 DNA ligase
+
<li> Ligase buffer</li>
-
5.      Distilled water
+
<li> T4 DNA ligase</li>
 +
<li>    Distilled water</li>
 +
</ol>
<br>
<br>
<h4 align="left">Prodecure:</h4>
<h4 align="left">Prodecure:</h4>
-
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>
+
<ol>
-
2. Add suitable amount of distilled water to make up total volume to 50 μL. <br>
+
<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>
-
3. Add 1 μL T4 DNA ligase buffer. <br>
+
<li> Add suitable amount of distilled water to make up total volume to 50 μL. </li>
-
4. Add 1 μL T4 DNA ligase. <br>
+
<li> Add 1 μL T4 DNA ligase buffer. </li>
-
5. Ligate at 16°C overnight or leave it at room temperature for 4 hours.  <br>  
+
<li> Add 1 μL T4 DNA ligase. </li>
 +
<li> Ligate at 16°C overnight or leave it at room temperature for 4 hours.  </li>
 +
</ol>
<br>
<br>
The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.
The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.

Revision as of 12:28, 29 September 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. Cut Insert DNA
  2. Cut Vector DNA
  3. Ligase buffer
  4. T4 DNA ligase
  5. Distilled water

Prodecure:

  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.
  2. Add suitable amount of distilled water to make up total volume to 50 μL.
  3. Add 1 μL T4 DNA ligase buffer.
  4. Add 1 μL T4 DNA ligase.
  5. Ligate at 16°C overnight or leave it at room temperature for 4 hours.

The next step would be to transform the new construct (approx ~ 1-2μL)into competent cells.

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