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

This protocol is based on QIAGEN® Plasmid Purification Handbook.
This protocol is designed for preparation of up to 20 µg of high-copy plasmid or cosmid DNA using the Qiagen Plasmid Mini Kit. Qiagen plasmid isolation is used to obtain very pure plasmid DNA.
Maximum recommended culture volumes for the Qiagen-tip 20:
High-copy plasmids 1-5 mL.

Requirements

1. bacterial culture
2. Qiagen colums
3. buffer P1 (100 μg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)
4. buffer P2 (200 mM NaOH, 1% SDS)
5. buffer P3 (3 M KAc, pH 5.5)
6. buffer PE
7. milliQ pH 8.0
8. centrifuge
9. nanodrop

Prodecure

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm).
2. Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
3. Resuspend pelleted bacterial cells in 250 μL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
4. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
5. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 mL) may require inverting up to 10 times. The solution should become cloudy.
6. Incubate at -20 °C for 15 minutes.
7. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.
8. Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting.
9. Centrifuge for 30–60 seconds. Discard the flow-through.
10. Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds.
11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
12. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 μL Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.
13. Measure DNA concentration on the Nanodrop.

References

1. Gel Extraction Kit ProtocolQIAquick Spin Handbook, Version 3, 2001.[Online]. Available From: http://devbio.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf