LAB9 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

LAB 9️⃣ PL3 Yeast DNA preparation ➕ E. coli transformation (II)

  • Prepare crude yeast DNA from cells with glass beads and an E. coli plasmid miniprep kif
  • Transform E. coli with crude yeast DNA
  • Plate E. coli on Petri dishes with solid LB-amp medium from LAB7

Plasmid preparation from S. cerevisiae, one per group

  1. (Scrape some yeast cells off a plate)
  2. (Add 1 mL water in a 2 mL Eppendorf tube)
  3. (Wash cells a and add glass beads)
  4. (Add 200 µl of glass beads (about one full 0.2 mL PCR tube) to a tube)
  5. (Add the amount of resuspension solution to the cells indicated by the kit)
  6. Vortex the tubes for 5 minutes using a disruptor genie.
  7. Add the lysis buffer as soon as possible, the disruption of the cells liberate nucleases that can damage the DNA.
  8. Follow the rest of the alkaline lysis mini-prep kit protocol. mb010_ifu_en_v2401.pdf.
  9. If the kit has an optional wash step to get rid of nucleases, use that.
  10. Elute in a small volume 50 µL or 30 µL for higher concentration.

E. coli transformations, one per group

  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. Incubate for up to 30 min on ice.
  3. Heat shock in water bath at 42°C during EXACTLY 45s.
  4. Cool the tube for 1-2 min in a water/ice slurry for fast heat transfer.
  5. Add 1 mL pre-warmed liquid LB medium to one and proceed to the next step or let cells recover at 37°C for 1 h if time allows.
  6. Pipette 300 µL of the content to a new Eppendorf tube. Give the remaining cells to the teacher.
  7. Add 20 µL ampicillin (x1000) and mix by inversion
  8. Pipette all of the content to a LB plate with 10-20 sterile glass beads and swirl the plate to spread the liquid.
  9. Incubate plates inverted for 18-24 h at 37°C

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