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

Operation method

Genetic technology of Chlamydomonas reinhardtii (transformation)

Materials and Instruments

Cells
EcoRI Enzyme
SGII medium

Move

1. 500 ml flasks were filled with 250 ml of SGII medium and cells were cultured to a density of ^5X106 /ml under aeration and constant light conditions.

2. Plasmids were digested with EcoRI.

3. Cell walls are removed, cells are concentrated by low-speed centrifugation, and resuspended to ¹⁰⁹ cells/ml in _SGII-NO3 medium.

4. Place the cells in a sterilized flask with the bottom of the flask just covered and shake gently under bright light for 4 hours.

5. For transformation, dilute the cells to 1. ^7X108 cells/ml and add 0.3 ml to a 15 ml conical centrifuge tube with a plastic cap containing 0.3 g glass beads.

6. Add sterilized PEG solution to a final concentration of 5%.

7. Immediately add 1 μg of linearized pMN24 DNA and spin for 45 seconds at maximum speed on a spin mixer.

8. Immediately add 10 ml of _SGII-NO3 medium dilution cross-mix, pour the cells into a clean 15 ml test tube and centrifuge the sediment in a tabletop medical centrifuge.

9. Reserve 0.3 ml of medium, resuspend the cells and spread on 1% agar plates in _SGII-NO3 medium.

10. Incubate the plate under constant light for 5-8 days.

11. Usually after 8 days of incubation after transformation, when the clones can be observed with the naked eye, use a sterilized toothpick to transfer individual clones to individual wells of a multiwell dish containing a small amount of _SGII-NO3 medium for overnight incubation.

12. Isolate the transformed pure clones by streaking them on plates of selective medium.

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