Team:TU-Delft/Protocol 6

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Revision as of 13:44, 27 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.