Rapid Yeast Transformation Kit
Protocol
Revision No. 2220-999-7D03W

Protocol

The Rapid Yeast Transformation Kit includes Uracil Deficient Yeast, a strain of yeast that requires uracil in its growth media to survive. This yeast will grow on YPD Agar plates since they include uracil and all other necessary nutrients. DOBA-URA Agar plates do not contain uracil, therefore the Uracil Deficient Yeast will not grow. However, after transforming the yeast cells with URA-plasmid DNA, the yeast will be able to survive on the DOBA-URA Agar plates because the plasmid contains the gene the yeast cells need to make uracil.

1. Yeast cells for this procedure are prepared 3-5 days before intended use by streaking a YPD Agar plate (start with step 2 if kit contains colonies on YPD Agar plates).



2. In a sterile microcentrifuge tube, suspend 3-4 single colonies in 200 ml of Lithium Cesium Acetate (cat. no. 2200-203).



3. Add 20 ml of Carrier Mixx with Plasmid (cat. no. 2220-201) to 200 µl of the cell suspension from step 2.



4. Add 1ml of PEG/TE/Cation Mixx (cat. no. 2220-204) to the tube and gently mix. Incubate for 20 minutes at room temperature. (The class should also treat cells without Carrier Mixx with Plasmid as a control; one or two tubes for the whole class are enough.)



5. Heat-shock (place tubes in 42°C water) cells for 10 minutes.



6. Centrifuge suspension for 1 minute in a microcentrifuge and quickly decant supernatant.



7. Resuspend pellet in 200 µl TE (cat. no. 2200-205).



8. Plate cells on agar plates containing uracil-selective media (DOBA-URA Agar, no. 2200-207) by spreading evenly over plate with spreader.



9. Incubate 3-5 days at room temperature.

Yeast transformants should be smooth white colonies although some colonies may have a pink tint. The number of transformants will range from 5 to 500 colonies per plate. Plates that were not treated with the Carrier Mixx with Plasmid should have few, if any, growing cells.