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Team:TU-Delft/Protocol 6
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Revision as of 15:28, 3 October 2013 by MaithiliKrishnan (Talk | contribs)
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:
- digested plasmid DNA or PCR product
- T4 ligation buffer (10x) (Fermentas)
- T4 ligase (Fermentas)
- H2O
- water bath at 16 °C
Procedure:
- Ligations (pasting 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 insert | x μL |
| DNA vector | x μL |
| T4 Ligation buffer (10×) | x μL (for 1×) |
| T4 Ligase | 1.0 μL |
| H2O | x μL |
| 10-15 μL |
- The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix.
Transform circa half of the ligation mix. Incubate at 16 °C o/n.
