Team:TU-Delft/Protocol 4

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
• Miniprep Protocol
• Restriction digestion
• Ligation
• Gel Extraction Procedure
• PCR Purification

Miniprep Protocol

We use Qiagen Spin Miniprep Kits for doing small batches of minipreps. This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.

Requirements :

1. Qiagen Miniprep Kit
2. Cell Culture
3. 1.6 ml Microcentrifuge tubes (2 per a miniprep)
4. TE (1:10)

Prodecure:

1. Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.
2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet. Important: Ensure that RNase A has been added to Buffer P1.
3. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. 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 min.
4. Add 350 μl Buffer N3 and invert the tube immediately and gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.
6. Apply the supernatants from step 5 to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 1 min.
9. 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. They are right about this.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

This plasmid DNA is stored in -20°C and is used to carry out restriction digestion when needed.