This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Electroporation transformation of heat-resistant tetrapods

Materials and Instruments

Antibiotic Resistant Cells
Tris Antibiotic Complex HEPES
Flasks Cormung plastic conical test tubes

Move

1. Cultivate appropriate antibiotic-resistant cells of 2 different mating types to logarithmic phase.

2. Wash cells at approximately ^2X105 cells/ml in 10 mmol/L pH 7.5 Tris and routinely mate to starve cells for 6-24 hours.

3. Determine the cell density of each mating type at ^3X105 cells/ml. mix equal amounts of cells of the opposite mating type in a flask of appropriate volume to ensure proper aeration during fixation.

4. add the antibiotic complex and cover the flask with aluminum foil or cotton wool stopper.

5. Begin mating and allow the cells to stand at 30 °C.

6. After 4-6 hours, check the mating efficiency. If the cells are more than 80% paired, proceed to the transformation step.

7. 10 hours after mating, transfer the cells to 50 ml Cormung plastic conical tubes and gently wash the cells with 10 mmol/L HEPES at pH 7.5.

8. Resuspend cells in 10 mmol/L HEPES to approximately ^3X107 cells/ml.

9. Mix 125 μl of cells and 125 μl of prepared plasmid DNA in HEPES buffer and electroporate immediately.

10. Use a 0.2 cm cuvette with the voltage set to 250 V, capacitance 275 mF and resistance 13 ohms.

11. The cuvette was allowed to stand for 1 minute at room temperature, and then the cells were resuspended in 20 ml of SPP with the antibiotic mixture added.

12. Dilute the cells with SPP series and add 25 ml to a 96-well microtitre plate containing 175 μl of SPP spiked with the antibiotic mixture.

13. 12-18 hours later, add 50 μl of Baronomycin, 600 μg/ml 10 mmol/L HEPES, or another antibiotic to each of the selected transformation wells.

14. After 4~5 days, the transformation yield was calculated using Poisson distribution.

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