Difference between revisions of "Team:KU Leuven/Research/Basic Part"

Protocol

1. Preparation of lysate starting from stock plate of phage

1. Make an overnight culture of E. coli MG1655.

2. Take 500 µL overnight culture and add the phage P1. Incubate overnight at 37°C.

3. Take single plaques of the P1 stock plate and bring this in a sterile Eppendorf tube together with 200 µL of mQ.

4. Overnight extraction while shaking at 37°C.

5. Add 0.01, 0.1, 10 and 100 µL of extraction to 500 µL of a stationary phase culture of E. coli MG1655. Vortex and plate out.

6. Add LB soft agar containing 10 mM MgSO₄ and 5 mM CaCl₂ and incubate at 37°C.

7. Choose the plate with the best lysates.

8. Sterilize your spoon using a Bunsen burner, cool it down with water and wash it with 100% ethanol.

9. Cut out a plaque from the soft agar and put this in a 10 mL syringe.

10. Press the content of the syringe in an Eppendorf tube and centrifuge for 10 minutes at 14000 rpm.

11. Take 650 µl and bring this in a new Eppendorf tube.

12. Extraction with 30 µL of CHCl₃.

13.Vortex vigorously.

14. Store lysate at 4°C.

2. Preparation of the lysate of donor strain

1. Firstly, centrifuge the lysate to ensure the chloroform is at the bottom of the Eppendorf tube. Then add 0.1, 1, 10 and 100 µl of lysate to 500 µL stationary phase overnight culture of the donor strain.

2. Add LB soft agar containing 10 mM MgSO₄ and 5 mM CaCl₂. Incubate this at 37°C.

3. Sterilize your spoon in a Bunsen flame, cool it down with water and wash with 100% ethanol.

4. Centrifuge the Eppendorf tubes 10 minutes at 14000 rpm.

5. Transfer 650 µL into a new Eppendorf tube.

6. Extract with 30 µL of CHCl₃.

7. Vortex vigorously.

8. Store the lysate at 4°C.

3. Transduction to acceptor strain

1. Concentrate 500 µL of stationary phase overnight acceptor strain culture five times in LB with 10 mM MgSO₄ and 5 mM CaCl₂.

2. Add 0.1, 1, 10 and 100 µL of donor strain lysate to 100 µL acceptor strain.

3. Incubate 30 minutes at 37°C.

4. Plate out on selective medium and incubate overnight.

5. Plate out lysate-only to check for contamination as well.

Theory
The Gibson assembly as described by Gibson et al., is a rapid DNA assembly method which assures directionional cloning of fragments in one single reaction. For the Gibson assembly to happen, three essential enzymes are needed: a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. For this reaction, we used NEBuilder. In the first step of this reaction, the exonuclease rapidly cleaves off the 5’ DNA ends. The exonuclease is unstable at 50°C and gets degraded. In the second step, the designed sequence overlaps anneal and the polymerase starts filling in the gaps. In the final step, the ligases covalently joins both ends. After this, the plasmid is ready to be transformed. This text was based on the IDT website as seen on 13/09/2015

Materials

Protocols

Protocol

1. Prepare selective media (LB with 0.25% agar (2,5 g/L) in Petri dishes (85 mm dia).

2. Apply 1.5 µL of the diluted cell suspensions from mid-log-phase cultures (~2×105 cells/µL (OD=0.5)) to the center of the plates, and let dry in air for 15 min.

3. Incubate at 37 °C for 10 h.