Transformation of Agro Using the Freeze-Thaw Method
- Thaw competent Agrobacterium on ice (use 250 ul per transformation reaction) and add DNA (1-5 ul of CsCl-purified DNA or 10 ul of standard E. coli miniprep DNA).
- Incubate the mixture on ice for 5 minutes
- Transfer mixture to liquid nitrogen and incubate for 5 minutes.
- Incubate the mixture for a further 5 minutes in a 37 degree C water bath.
- Add 1 ml of LB to each tube, seal well, and place the tubes on a rocking table for 2-4 hours at room temperature.
- Collect the cells by spinning briefly in a microcentrifuge and spread them on two LB agar plates containing the appropriate antibiotic. Include the antibiotic for the T-DNA vector.
- Incubate cells for 2 days at 28 degrees C.