Team:TU-Delft/Protocol 5

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:

1. plasmid DNA or PCR product
2. restriction enzymes (Roche and BioLabs)
3. buffer (10x)
4. H2O
5. water bath at 37 °C
6. heat block or water bath at 65 °C

Prodecure:

1. Digestions (cutting 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:
Table 1: Lysis experiment

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.