Team:TU-Delft/Protocol 7

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

Gel Extraction Procedure

Requirements:

1. Scalpel
2. Pipettes
3. Microcentrifuge tubes
4. QIA quick column and collection tubes
5. Buffer PE
6. Buffer QG
7. Sterile MilliQ

Prodecure:

1. Excise the DNA fragment from the agarose gel with a clean scalpel.
2. Weigh the gel slice in a tube. Add 3 volumes of Buffer QG to 1 volume of the gel.
3. Incubate at 50 ⁰C for 10 mins (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 mins during incubation.
4. After the gel slice has dissolved completely check that the colour of the mixture is yellow.
5. Place a QIA quick spin column in 2mL collection tube and centrifuge for 1 min.
6. Discard the flow through and place QIA quick column back in same collection tube.
7. To wash, add 0.75 mL of Buffer PE to the QIA quick column and centrifuge for 1 min at 13000 rpm.
8. Discard the flow through and centrifuge for an additional 1 min at 13000 rpm to remove the remaining ethanol.
9. Place the column on a clean microcentrifuge tube.
10. Add 40-50 µL of milliQ sterile H2O.
11. Incubate in oven for 2 mins and then centrifuge again for 1 min.
12. Measure on Nanodrop for the concentration of the DNA.