Lissa1:Yeast Transformations

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Day 1.

  1. Inoculate the yeast strain into 5 mL of SC selection medium and incubate overnight on a rotary shaker at 200 rpm (but 230 is fine) at 30 degrees.

Day 2.

  1. Get the OD600 of your yeast. If it looks like the reading will be over an OD of 1, do a 1:10 dilution and multiply the OD600 reading by 10.
  2. Use this to figure out how many cells you have per mL. If your OD is .1, you have 10^6 cells/mL.
  3. Transfer 50 mL of 1x YPD in to a culture flask and add 2.5*10^8 cells (figure out the volume from step b) to make a final dilution of 5*10^6 cells/mL.
  4. Incubate your flask on a rotary shaker at 200 rpm at 30 degrees until the cells have divided at least twice (you’ll know because the OD will double twice). It takes about 3-5 hours.
  5. When the OD is 2*10^7 (or it’s doubled), harvest cells by centrifuging at 3000g for 5 min (this is a setting of 2.5 on the big centrifuge). You need a large centrifuge tube for this.
  6. Wash your cells in 25 mL of sterile water, and centrifuge again, same as before.
  7. Resuspend in 1 mL of sterile water.
  8. Boil a 1 mL sample of carrier DNA (salmon sperm in our case) for 5 minutes and chill on ice…. Unless this has already been done in which case the carrier is in the -20 freezer.
  9. Transfer the cell suspension to a 1.5 mL eppendorf tube and microfuge for 30 sec. Discard the supernatant.
  10. Add water to a final volume of 1 mL and vortex to resuspend. You may need to use a different volume depending on your OD.
  11. Pipette 100 uL samples in to eppendorfs (1 per transformation), microfuge at top speed for 30 seconds and discard supernatant.
  12. Make up Transformation Mixture.
    1. Each tube will get:
      1. 240 uL PEG 3500 50% w/v
      2. 36 uL LiAc 1.0 M
      3. 50 uL boiled SS-carrier DNA
      4. 34 uL plasmid DNA plus water
        1. total = 360 uL
    2. If you are doing 5 transformations, here’s what you do:
        1. A master mix of:
          1. 1440 uL PEG
          2. 216 uL LiAc
          3. 300 uL carrier DNA
    3. Put 326 uL of the master mix in to a tube for each of your transformations. In to the tubes you can add the appropriate plasmid/water mix to get the last 34 uL.
  13. VORTEX.
  14. Incubate in a 42 degree water bath for 40 mins.
  15. Microfuge at 13 for 30 seconds and remove supernatant.
  16. Pipette 200 uL sterile water in to each tube, stir pellet with tip and vortex.
  17. Plate appropriate amount of cell suspension on to SC selection medium.
  18. Incubate at 30 degrees for 3 or 4 days.
  19. Hope that it works!
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