Product Data Sheet

Direct chemical transformation of Agrobacterium AGL-1:

Freeze-thaw method

1. Add about 1 μg of plasmid DNA to the cells.

2. Freeze the cells in liquid nitrogen.

3. Thaw the cells by incubating the test tubes in a 37°C water bath for 5 min.

4. Add 1 ml of YEP 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.

5. Centrifuge the tubes for 30s in an Eppendorf centrifuge. Discard the supernatant solution. Resuspend the cells in 0.1 ml YEP medium.

6. Spread the cells on a YEP agar plate containing appropriate antibiotic selection. Incubate the plate at 28°C. Transformed colonies should appear in 1-3 days.

Storage of Agrobacterium Strains by Low-Temperature Freezing

1. Inoculate 2 or 3 mL of YEB media that contains the appropriate antibiotics for plasmid maintenance with your strain).

2. Grow the culture to early or middle stationary phase (usually overnight). Chill the culture and the sterile medium containing 50% glycerol on ice.

3. Add an equal volume of the medium with glycerol to make the culture 25% glycerol. Mix culture and medium completely.

4. Transfer the cells to a labeled 2-mL tube, and place it in the freezer. The frozen culture can be stored indefinitely.

5. To recover the strain, simply scrape a bit of the frozen culture onto plate medium

containing the appropriate antibiotics. This can be done with a sterile, cooled,

inoculating loop or with a sterile pipet tip. It is important to handle the culture

quickly to prevent thawing and refreezing. If handled properly, the same culture

can used repeatedly for retrieving the bacteria strain.