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
- (Scrape some yeast cells off a plate)
- (Add 1 mL water in a 2 mL Eppendorf tube)
- (Wash cells a and add glass beads)
- (Add 200 µl of glass beads (about one full 0.2 mL PCR tube) to a tube)
- (Add the amount of resuspension solution to the cells indicated by the kit)
- Vortex the tubes for 5 minutes using a disruptor genie.
- Add the lysis buffer as soon as possible, the disruption of the cells liberate nucleases that can damage the DNA.
- Follow the rest of the alkaline lysis mini-prep kit protocol. mb010_ifu_en_v2401.pdf.
- If the kit has an optional wash step to get rid of nucleases, use that.
- Elute in a small volume 50 µL or 30 µL for higher concentration.
E. coli transformations, one per group
- 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.
- Incubate for up to 30 min on ice.
- Heat shock in water bath at 42°C during EXACTLY 45s.
- Cool the tube for 1-2 min in a water/ice slurry for fast heat transfer.
- 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.
- Pipette 300 µL of the content to a new Eppendorf tube. Give the remaining cells to the teacher.
- Add 20 µL ampicillin (x1000) and mix by inversion
- Pipette all of the content to a LB plate with 10-20 sterile glass beads and swirl the plate to spread the liquid.
- Incubate plates inverted for 18-24 h at 37°C
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