Yeast Transformation

Low Efficiency (suitable for episomal plasmid)

• grow yeast on YPD plate overnight
•  resuspend in buffer

100ul of buffer100ul 2M Lithium Acetate (LiAc)
100ul 1M DDT
800ul 50%PEG (3350 or 4000)
50ul ssDNA (10mg/ml)

• add DNA (50ng+)

• vortex
• heat shock 42C 14min
• plate on selective media and wait

High Efficiency (more than one plasmid, integrative plasmids, endogenous tagging)

Competent cells preparation:

• grow yeast in 5ml YPD overnight
  
• dilute in 30ml to 0.1-0.2 OD
  
• grow to 0.5-0.8 OD ~3h
• spin cells (3000rpm 5min), wash ddW (~20ml)
• spin cells, wash with 0.1M LiAc
  
• spin cells, discard (there will be some LiAc left for ~5 transformations
• keep in +4C up to 1week

Transformation:

• prepare mix: 

480ul PEG50%
100ul competent cells
72ul 1M LiAc
40ul ssDNA (10mg/ml)
DNA (plasmid/PCR product...)

• vortex
  
• 30min-1h 30C without rotation
  
• add 72ul DMSO, vortex
  
• heatshock 42C 14min
  
• spin cells, discard, resuspend in 100ul of water and plate on selective media, if you’re working with antibiotic resistance markers plate on YPD, next day replate on selective media

Yeast long term storage

• Grow yeast colony overnight in YPD (4ml)
  
• Transfer cells to sterile tube in YPD or selective media, add glycerol to 15%
  
• Freeze -80C
  
• To recover cells plate them on YPD by scraping (do not thaw the stock!)