Team:TU-Delft/Protocol 6

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:

1. digested plasmid DNA or PCR product
2. T4 ligation buffer (10x) (Fermentas)
3. T4 ligase (Fermentas)
4. H2O
5. water bath at 16 °C

Procedure:

1. Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
2. 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.