Yeast Electoporation Transformation OLD!!

Electroporation of yeast cells, based on protocol by Pernilla Bjerling

This is an Old protocol. For the new version which includes the option to make cryopreservation see Yeast Electoporation Transformation

• Grow the strain in YES medium to density of approximately 1 X 10⁷ (Generation time of approx 2.5 h @32°C. For 5 transformations grow 100 ml)
• Pre-cool enough cuvettes in the fridge and make sure there is enough sterile Sorbitol 1M.
• Spin down the cells at 4500 rcf at 4°C for 10 min, remove supernatant and put on ice.
• Wash by adding 20 ml of ice-cold 1M Sorbitol, resuspend cells and spin down again.
• Wash in total 3 times and when using more than 1 falcon-tube per strain merge tubes together during washing.
• Clean the PCR product or plasmid to remove all salts by column or dialysis to a concentration of 0.1-1 μg in max 20 μl.
• Dilute the cell suspension to a density of 1 X 10⁹ cells/ml with 1M Sorbitol.
• Divide the cell dilution to pre-chilled Eppendorf-tubes with a volume of approx. 200 µl.
• Prepare everything at the electroporation equipment to do efficient transformations.
• Add 0.1-1 μg DNA (maximum of 20µl, but fewer is better) to be integrated to the cells and immediately transfer the cell + DNA mixture to a pre-chilled electroporation chamber.
• Transform using 2 mm electroporation chambers and the settings 2250 V. Change the settings for resistance to 200Ω and capacitor to 25µF.
• Always make a negative control without DNA as well!!
• After electroporation, immediately add 500 µl of fresh 1 M Sorbitol to the cells and transfer cells to an Eppendorf tube and place on ice.
• Continue with the rest of the transformations.
• Plate out each transformation on non-selective YES plates, spread with beads or spatula and let them dry.
• After 16-20h incubation at 32°C, replica plate to selective plates (YES + G418).
• Incubate until colonies are clearly visible.
• Pick several (large) colonies from the selective plates and transfer to YES+G418
• Re-streak on YES plates grow for 2 days and restreak again. Repeat this 2 more times (total 1x YES-G418, 3x YES). Cells without integration will lose non-integrated fragments and become G418 sensitive.
• Streak out on YES to get separate colonies.
• Replicate plate onto YES-G418 and onto YES. If all colonies grow on both plates, the transformant is stable.
• Use colony PCR to test for integration using primers annealing at 5` and 3` of the gene or insertion site.