Team:TU-Delft/Protocol 5

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<h2 align="center">Restriction digestion</h2>
<h2 align="center">Restriction digestion</h2>
<h4 align="left">Requirements:</h4>
<h4 align="left">Requirements:</h4>
-
1. Plasmid DNA
+
<ol>
-
2. Restriction Enzymes
+
<li> Plasmid DNA</li>
-
3. 1.5 mL Tubes
+
<li> Restriction Enzymes</li>
-
4.      NE Buffer 2
+
<li> 1.5 mL Tubes</li>
-
5.      BSA
+
<li>    NE Buffer 2</li>
-
6.     Distilled water
+
<li>    BSA</li>
 +
<li>     Distilled water</li>
 +
</ol>
<br>
<br>
<h4 align="left">Prodecure:</h4>
<h4 align="left">Prodecure:</h4>
-
1. 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. <br>
+
<ol>
-
2. Add 5 μL NE Buffer 2 and 0.5 μL  of BSA. <br>
+
<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>
-
3. There should be a total volume of 50 μL. Mix well and spin down briefly.<br>
+
<li> Add 5 μL NE Buffer 2 and 0.5 μL  of BSA. </li>
-
4. 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. <br>
+
<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>
 +
</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.
Run a portion of the digest on a gel (8ul, 100ng)to check that both plasmid backbone and part length are accurate.

Revision as of 12:26, 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.


Restriction digestion

Requirements:

  1. Plasmid DNA
  2. Restriction Enzymes
  3. 1.5 mL Tubes
  4. NE Buffer 2
  5. BSA
  6. Distilled water

Prodecure:

  1. 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.
  2. Add 5 μL NE Buffer 2 and 0.5 μL of BSA.
  3. There should be a total volume of 50 μL. Mix well and spin down briefly.
  4. 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.

Run a portion of the digest on a gel (8ul, 100ng)to check that both plasmid backbone and part length are accurate. The next step would be to carry out ligation of the digested pieces of DNA.

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