Team:TU-Delft/Protocol 8

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

PCR Purification:

Requirements:

1. Buffer PB
2. Pipettes
3. Buffer PE
4. Microcentrifuge tubes
5. QIA quick spin columns

Procedure:

1. Add 5 volumes of Buffer PB to 1 volume of the sample.
2. Transfer the mixture to the QIA quick spin columns and centrifuge the column for 1 min at 13000 rpm.
3. Discard the flow through and place the spin column in the same collection tube.
4. To wash, add 0.75 mL of Buffer PE to the column, and centrifuge for 1 min. Discard the flow through and place the column back in the same collection tube.
5. Centrifuge the empty column for an additional 1 min to remove the remaining Buffer PE from the column.
6. Now place the column in a fresh microcentrifuge tube. Add 50 µL of sterile milliQ water.
7. Place the column in oven for 2 mins, then centrifuge again for 1 min at 13000 rpm.
8. Measure the sample on Nanodrop to get the concentration.



References

1. PCR Purification Kit ProtocolQIAquick Spin Handbook, July 2002 .[Online]. Available From: http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf