Yeast transformation

From: "High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method". R Daniel Gietz & Robert H Schiestl, Nature Protocols 2, 31 - 34 (2007) Published online: 31 January 2007

(Added by Laura Bassolino and Elena Loreti)

  • Inoculate a single colony of the yeast strain with sterile inoculation loop from a fresh YAPD plate into 5 mL of liquid culture medium and incubate O/N on a rotary shaker at 180-200 rpm at 30°C
  • After 12-16h of growth determine the titer of the yeast culture using spectrophotometer. Pipette 10 mL of yeast culture into 1 mL of water mix and measure the OD at 600nm (a suspension containing 1x106 cell mL-1 give an OD 600 of 0.1). Remember to multiply by the diluition factor to determine the titer in  the culture
  • Add 40 mL of  YAPD
  • Incubate the flask (falcon tube) in the shaking incubator at 30°C at 200 rpm for 3-4 hours
  • Centrifuge the culture at 5000 rpm for 5’ at 22°C and resuspend the pellet in 25 sterile water and centrifuge at 5000 rpm for 5’ at 22°C
  • Repeat this wash with another 25 mL of sterile water by resuspending the cells and pelleting them again by centrifugation. Finally resuspend the cells in 1 mL of sterile water
  • Meanwhile denature a 1 mL sample of carrier DNA in a boiling water bath for 5’ and chill immediately in ice
  • Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge for 30s at 10000 rpm and discard the supernatant
  • Resuspend the cells in 1 mL of sterile water and pipette 100 mL samples into a 1.5 mL microcentriguge tubes, one for each transformation. Centrifuge for 30s at 10000 rpm and remove the supernatant
  • Meanwhile prepare the transformation mix. Add 336 mL of transformation mix to each transformation tube containing the cell pellet. Add the plasmid DNA (1600ng in total) to each of the tubes plus water to a final volume of 34 mL. Always include a negative control tube containing no plasmid DNA
  • Resuspend the cell pellet by vortex
  • Place the tube at 42°C for 30’. Each strain may have different optimum heat-shock time
  • Centrifuge the tube at 10000 rpm for 30s,  remove the supernatant and pipette 1 mL of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break the cell pellet and then vortex
  • Plate 50, 100 mL of the cell suspension onto the appropriate SC selection medium. The volume plated will depend on the efficiency of your yeast strain. Allow the liquid to be adsorbed into the medium by incubation at room temperature
  • Incubate the plates at 30°C for 2-4 days 

 

Transformation Mix:

PEG 3350 (50% w/v) 240 µl

LiAc 1M 36 µl

Single-stranded carrier DNA (2 mg mL-1) 50 µl

Total volume: 326 µl