Maheshri:TransformationY

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The TRAFO method

This protocol can be used to generate sufficient transformants in a single reaction to screen multiple yeast genome equivalents for plasmids that complement a specific mutation. It can also be used to transform integrating plasmids, DNA fragments and oligonucleotides for yeast genome manipulation. Finally, it is used to optimise the conditions for transformation of a particular yeast strain, for example, the transformation of a plasmid library into a two-hybrid yeast strain transformed with a bait plasmid by the Rapid Transformation Protocol. The High Efficiency Protocol can also be employed to transform a yeast strain simultaneously with two different plasmids, such as the two-hybrid bait and prey plasmids.

Reagents

Single-stranded Carrier DNA (2 mg/ml)

  1. Weigh out 200 mg of high molecular weight DNA (Deoxyribonucleic acid Sodium Salt Type III from Salmon Testes, Sigma D1626) into 100 mL of TE buffer (10 mM Tris-HCl pH 8.0, 1.0 mM EDTA).
  2. Disperse the DNA into solution by drawing it up and down repeatedly in a 10 mL pipet.
  3. Mix vigorously on a magnetic stirrer until fully dissolved.
  4. Aliquot the DNA and store in a -20°C freezer.
  5. Prior to use, an aliquot should be placed in a boiling water bath (or a heat block set to 120 °C) for at least 5 min and quickly cooled in an ice water slurry.

Lithium Acetate Stock Solution (1M)

  1. The lithium acetate solution is prepared as a 1.0 M stock in ddH2O and filter sterilized.

Polyethylene glycol (PEG 50% w/v)

For 10mL:

  1. Place 5 g of polyethylene glycol, MW 3350 (Sigma) in a 15 mL Falcon tube and add 3mL of ddH20.
  2. Microwave for 10-15 seconds, or until all powder is dissolved. Mix by vortexing vigorously.
  3. QS to 10 mL using the marks on the Falcon tube. Mix well by vortexing.
  4. Filter sterilize using a 0.45 µm filter and a 10mL syringe.

Note:

  • For optimal transformation efficiencies, care must be taken to ensure that the PEG solution is at the proper concentration. Store the PEG in the 4°C refrigerator.

Full Protocol

Day 1

Inoculate the yeast strain into 2-5 mL of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of 2x YPAD and a 250 ml culture flask in the incubator as well.

Day 2

  1. Determine the OD of the yeast culture. Dilute the overnight YPAD or SC cultures in 50mL of 2x YPAD to an OD of ~0.1.
  2. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm. Note that:
    • It is important to allow the cells to complete at least two divisions.
    • This will take 3 to 5 hours.
    • This culture will give sufficient cells for 10 transformations.
    • Transformation efficiency (remains constant for 3 to 4 cell divisions.
  3. When the OD becomes ~1, which should take about 5 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 mL of sterile water and resuspend in 1 ml of sterile water.
  4. While your are harvesting the cells, boil a 1.0 mL sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells. Note that it is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.
  5. Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
  6. Add water to a final volume of 1.0 mL and resuspend the cells by pipetting. If the OD of the culture is greater than 1 (but not much greater) then increase the volume accordingly. If the OD is less than 1 then decrease volume.
  7. Pipette 100 µL samples (~108 cells) into 1.5 mL microfuge tubes, one for each transformation, centrifuge at 3000 min for 1 min and remove the supernatant.
  8. Add the following Transformation Mix to each tube, resuspend the cells by pipetting and gentle vortexing (speed 7).
    • PEG 3500 50% w/v 240 µL
    • LiAc 1.0 M 36 µL
    • Boiled SS-carrier DNA 50 µL
    • Plasmid DNA plus Water 34 µL
    • Total 360 µL
  9. Incubate the tubes in a 42°C water bath for 40 min. Note that the optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.
  10. Microcentrifuge at 3000 rpm for 1 min and remove the Transformation Mix with a micropipettor.
  11. Optional - Wash (resuspend and pellet) the cells with 1.0 mL of sterile water
  12. Add 200μL of sterile water to the tube, resuspend cells, and plate the cell suspension onto selection medium (e.g. a SD dropout plate).

A 'sleazy' protocol for plasmid transformation

  1. Using sterile technique, mix the following in a microcentrifuge tube:
    • PEG 3500 50% W/V 240μL
    • LiAc 1.0M 36μL
    • Boiled SS-carrier DNA 50μL
    • Plasmid DNA plus ddH2O 34μL
    • A toothpick full of yeast from a fresh plate (<2 days old)
  2. Incubate the tubes at room temperature for overnight (>8 hours)
  3. Microcentrifuge at 3000rpm for 1 min and remove the Transformation Mix
  4. Optional - Wash (resuspend and pellet) the cells with 1.0 mL of sterile water
  5. Add 200μL of sterile water to the tube, resuspend cells, and plate the cell suspension onto selection medium (e.g. a SD dropout plate).
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