Quint Lab:yeast transformation

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yeast transformation - quick and easy

  1. Inoculate 2 ml over night culture (selective medium) (OD 0.6 –0.8 is better, maybe dilute and let grow again for few hours in the morning)
  2. Spin down slowly
  3. Resuspend in 100 mM LiAc
  4. Incubate 45 min (or longer) @30°C
  5. Place the volume representing a single 20 . 50 µl inoculum into a separate microcentrifuge tube
  6. Add the following components into the tube on top in this order:
    1. 240 µl PEG (50% w/v)
    2. 36 µl 1.0 M LiAc
    3. 50 µl ss-DNA (2.0 mg/ml) (just 5 min boiled before and cooled on ice-water)
    4. 5.0 – 30 µl plasmid DNA (100 ng to 5 µg)
  7. Vortex 30 s to mix and incubate @ 42°C for 20 min (or longer)
  8. Pellet the cells at top speed in a microcentrifuge for 10 – 30 s
  9. Remove SN using a micropipet
  10. Gently resuspend the pellet in 50 µl of MQ-water by slowly pipetting up and down
  11. Plate the cell suspension onto a plate of SC ommision medium that selects for the presence of the plasmid
  12. Colonies should be visible in 2 –3 days @ 30°C
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