Yeast Electoporation Transformation OLD!!
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Electroporation of yeast cells, based on protocol by Pernilla Bjerling
NOTE: 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.