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Team:TU-Delft/Protocol 6
From 2013.igem.org
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.
- Transforming Parts from Distribution kit
- Growing the Single Colonies from the Agar Plates
- Making glycerol stocks
- Plasmid Purification Protocol
- Restriction digestion
- Ligation
- Gel Extraction Procedure
- PCR Purification
- Tricine Gels
- General Peptide Production
- SUMO cleavage
- Lysis Protocol
- AIP Sensing Protocol
- Gene Design
- Primer Design
Ligation
Requirements:
- Cut Insert DNA
- Cut Vector DNA
- Ligase buffer
- T4 DNA ligase
- Distilled water
Prodecure:
- 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.
- Add suitable amount of distilled water to make up total volume to 50 μL.
- Add 1 μL T4 DNA ligase buffer.
- Add 1 μL T4 DNA ligase.
- 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.
