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Team:TU-Delft/Protocol 5
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
Restriction digestion
Requirements:
- plasmid DNA or PCR product
- restriction enzymes (Roche and BioLabs)
- buffer (10x)
- H2O
- water bath at 37 °C
- heat block or water bath at 65 °C
Procedure:
- Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
- Reaction for one sample:
| Component | Sample |
| DNA | x μL (up to 1,0 μg) |
| Buffer (10x) | x μL (for 1×) |
| Restriction enzymes | x μL (10 units/μg DNA = 1 µL) |
| H2O | x μL |
| 20-25 μL |
- Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C.
- Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.
