BMBMethod:AgroTransform

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Agrobacterium Chemical Competent Cells and Transformation

Once a desired molecule is constructed in E. coli, the molecule can be transferred into Agrobacterium by the freeze-thaw method. Although the transformation frequency by these methods is low (approximately 10³ transformants per µg DNA) compared to the triparental mating method, the technique is reliable and very rapid. This transformation procedure also eliminates much of the plasmid rearrangement that often occurs during triparental mating.
Steps in the procedure:

1. Grow an Agrobacterium strain containing an appropriate helper Ti plasmid in 5 ml of YEP or LB medium overnight at 28°C.
2. Add 2 ml of the overnight culture to 50 ml YEP medium in a 250-ml flask and shake vigorously (250 rpm) at 28°C until the culture grows to an OD₆₀₀ of 0.5 to 1.0.
3. Chill the culture on ice. Centrifuge the cell suspension at 3000g for 5 min at 4°C.
4. Discard the supernatant solution. Resuspend the cells in 1 ml of 20 mM CaCl₂ solution (ice-cold). Dispense 0.1-ml aliquots into prechilled Eppendorf tubes.
5. Add about 1 µg of plasmid DNA to the cells.
6. Freeze the cells in liquid nitrogen.
7. Thaw the cells by incubating the test tubes in a 37°C water bath for 5 min.
8. Add 1 ml of YEP or LB medium to the tube and incubate at 28°C for 2-4 h with gentle shaking. This period allows the bacteria to express the antibiotic resistance genes.
9. Centrifuge the tubes for 30s in an microfuge. Discard the supernatant solution. Resuspend the cells in 0.1 ml YEP medium.
10. Spread the cells on a YEP or LB agar plate containing appropriate antibiotic selection. Incubate the plate at 28°C. Transformed colonies should appear in 2-3 days.

Notes

• The cells can be frozen in liquid nitrogen and stored at -80°C. The frozen cells can be used for future transformation experiments. Add about 1 µg of DNA to the frozen cells and follow the steps 7-10.
• Quick freezing is the most important factor of the procedure. Freezing in a dry ice/ethanol bath often reduces the transformation frequency significantly.

References

An et al. Plant Molecular Biology Manual, 1-19 (1988).