LAB7️⃣ PL3 plasmid rescue, E. coli transformation - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki
- 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 LAB6
Pour two mL of yeast culture into a 2 mL Eppendorf tube.
Spin for 20 s in a microcentrifuge
Add the amount of resuspension solution to the cells indicated by the kit.
Resuspend cells by vortexing briefly.
Add 200 µl of glass beads (about one full 0.2 mL PCR tube).
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. .
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.
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 30 min on ice.
Heat shock in water bath at 42°C during EXACTLY 45s.
Cool the tube for 2 min in a water/ice slurry for fast heat transfer.
Add 1 mL pre-warmed liquid LB medium to one and let cells recover at 37°C for 1 h.
Centrifuge @ ~5000 rpm for 2-3 min.
Discard ~900 µL of the supernatant, so that 200-300 µL remains
Resuspend pellet in the remaining liquid.
Plate by adding 10-20 sterile glass beads to an LB ampicillin and swirl the plate to spread the liquid.
