Maheshri:TransformationY

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: Note:
 * 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.
 * 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:
 * 3) *It is important to allow the cells to complete at least two divisions.
 * 4) *This will take 3 to 5 hours.
 * 5) *This culture will give sufficient cells for 10 transformations.
 * 6) *Transformation efficiency (remains constant for 3 to 4 cell divisions.
 * 7) 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.
 * 8) 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.
 * 9) Transfer the cell suspension to a 1.5 mL microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
 * 10) 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.
 * 11) 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.
 * 12) Add the following Transformation Mix to each tube, resuspend the cells by pipetting and gentle vortexing (speed 7).
 * 13) *PEG 3500 50% w/v          240 µL
 * 14) *LiAc 1.0 M                36 µL
 * 15) *Boiled SS-carrier DNA     50 µL
 * 16) *Plasmid DNA plus Water    34 µL
 * 17) *Total                     360 µL
 * 18) 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.
 * 19) Microcentrifuge at 3000 rpm for 1 min and remove the Transformation Mix with a micropipettor.
 * 20) Optional - Wash (resuspend and pellet) the cells with 1.0 mL of sterile water
 * 21) 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:
 * 2) *PEG 3500 50% W/V       240μL
 * 3) *LiAc 1.0M              36μL
 * 4) *Boiled SS-carrier DNA  50μL
 * 5) *Plasmid DNA plus ddH2O 34μL
 * 6) *A toothpick full of yeast from a fresh plate (<2 days old)
 * 7) Incubate the tubes at room temperature for overnight (>8 hours)
 * 8) Microcentrifuge at 3000rpm for 1 min and remove the Transformation Mix
 * 9) Optional - Wash (resuspend and pellet) the cells with 1.0 mL of sterile water
 * 10) Add 200μL of sterile water to the tube, resuspend cells, and plate the cell suspension onto selection medium (e.g. a SD dropout plate).