Transforming yeast

Creating competent yeast cells

1. Grow yeast in 250 ml YEPD until A600 is 0.2-0.8. There are several ways to do this. For maximum efficiency, it is important that the yeast plate used to inoculate the YEPD is not too old, and that the yeast is growing well in log phase. Approach 3 below is the best approach for high efficientcy. In a test transformation, yeast grown at 30C became more competent than yeast grown at 20C, though this should be confirmed. o Inoculate the YEPD with a patch of yeast in the afternoon and grow O/N at room temperature. o Inoculate the YEPD with an overnight culture to A600 of ~0.1 and grow at 30C to A600 of 0.5-0.8. o Inoculate the YEPD late in the day, grow O/N so the A600 reaches ~0.1. Continue growing at 30C to A600 of 0.5-0.8. 2. Pellet the yeast at 1800 rpm, 20C for 3 min. 3. Resuspend in 50ml sterile H2O at RT. 4. Pellet the yeast at 1800 rpm, 20C for 5 min. 5. Resuspend in 50ml TE/LiAc at RT. 6. Spin again. 7. Resuspend in TE/LiAc in a total volume of 0.5 ml per 0.1 A600.

Small scale transformation

Before starting the transformation, boil Salmon sperm DNA for 5 minutes and immediately put in an ice water bath to prevent the strands from reannealing. 1. For each transformation mix the following. The easiest is to prepare a master mix of all the components except for the DNA, adding the yeast last so as not to shock it with a high concentration of LiAc. Prepare the mix just before adding the DNA so the Salmon sperm DNA stays single stranded. Component Competent yeast Salmon sperm DNA (2mg/ml) 10x TE 1M LiAc 60% PEG [MW3,350] Plasmid (200 ng) in water Final Volume 2. 3. Incubate at 30C for 30 min. 4. Incubate at 42C for 15 min. 5. Add 1 ml H2O. Amount in l 20 20 11 13 82 4 150

6. Spin briefly in a microfuge, and resuspend in 20l H2O to spot or 200l H2O to plate.

Large scale transformation

This protocol is for 30 g of DNA to be plated on 30 large plates, but can easily be scaled up. Before starting the transformation, boil Salmon sperm DNA for 5 minutes. 1. In a 15ml tube, mix the following, adding the yeast last so as not to shock it with a high concentration of LiAc. Prepare the mix just before adding the DNA so the Salmon sperm DNA stays single stranded. Component Competent yeast Salmon sperm DNA (2mg/ml) 10x TE 1M LiAc 60% PEG [MW3,350] Plasmid (30 g) in water Final Volume 2. 3. 4. 5. 6. 7. 8. Amount in l 1600 1600 880 1040 6560 320 12 ml

Aliquot the 12 ml into 1 ml eppendorf tubes. Incubate all eppendorf tubes at 30C for 30 min. Incubate all eppendorf tubes at 42C for 15 min. Spin in microfuge for 5 sec. Resuspend pellets in 750 l H2O and pool the yeast (9 ml total volume). Add 10 l of yeast to 10 ml H2O and plate 300 l of this as a transformation control (efficiency = nr. of colonies X 30,000). 9. Plate 300 l of the final mixture onto 30 plates with appropriate selective markers.