LAB8 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

LAB 8️⃣ PL3 Yeast DNA preparation ➕ E. coli transformation

material#LAB8 Summary:

  • Prepare crude yeast DNA from cultures prepared in LAB7.
  • Transform E. coli with crude yeast DNA.
  • Plate E. coli on Petri dishes with solid LB medium from LAB7

Plasmid preparation from S. cerevisiae, one per student

  1. Each student has at least one Eppendorf tube with S. cerevisiae frozen cells from LAB7.
  2. Add 200 µL P1 solution to the frozen cells
  3. Add 200 µl of glass beads (about one full 0.2 mL PCR tube).
  4. Resuspend cells by vortexing briefly.
  5. Vortex the tubes for 5 minutes using a disruptor genie.
  6. Add 200 µL of P2 solution (use googles, solution has NaOH). Do this as soon as possible, the disruption of the cells liberate nucleases that can damage DNA.
  7. Invert tube slowly for 3 min.
  8. Add 200 µL P3 solution.
  9. Invert tube slowly for 3 min.
  10. Spin (centrifuge) tubes for 10 min at highest speed.
  11. While the tubes are spinning, prepare one 1.5 mL Eppendorf tube with 1 mL 96-100% ethanol
  12. Transfer 400-500 µL of the supernatant from the centrifugation to the tube with ethanol.
  13. Invert tube slowly ~10 times to mix.
  14. Spin (centrifuge) tubes for 10 min at highest speed.
  15. Remove all liquid by decanting.
  16. Add 1 mL 70% ethanol to the tube
  17. Spin (centrifuge) tubes for 1 min at highest speed.
  18. Remove all liquid by decanting.
  19. Remove as much liquid as possible with a pipette, spin again if necessary.
  20. Dry the DNA for 5 min at 50°C.
  21. Add 50 µL of TE buffer to the precipitated DNA and vortex briefly to dissolve.
  22. Label your tube with the number in the Google sheet next to your name.

E. coli transformations, one per student

  1. Add the 10 µL of the plasmid DNA to the tube with competent cells, flick the tube a few times to mix. Do NOT vortex the cells at this point.
  2. prepare a cup with ice/water slurry.
  3. Incubate for 5-10 min on ice.
  4. Heat shock in water bath at 42°C during EXACTLY 45 s ⏱️.
  5. Cool the tube for 2 min in a water/ice slurry for fast heat transfer.
  6. Add 300 µL pre-warmed (37°C) liquid LB medium.
  7. Add 20 µL ampicillin to the cells.
  8. Mix cells by pipetting slowly up and down.
  9. Plate 200 µL by adding 20 - 30 sterile glass beads to an LB plate and swirl to spread the cells.
  10. Incubate inverted at 37°C for 18 - 24 hours. This protocol is described in greater detail here. colored.png
⚠️ **GitHub.com Fallback** ⚠️