Meskipun isolasi plasmid dari sel Agrobakteria jarang dilakukan, namun beberapa peneliti masih melakukannya. Tujuannya sama dengan isolasi DNA dari E. coli, yaitu untuk konfirmasi bahwa transformasi dari gen yang kita inginkan telah berhasil masuk ke sel Agrobakteria. Langkah-langkahnya adalah sebagai berikut:
- Grow Agrobacterium cells overnight in 1 mL YEP media at 28°C with shaking.
- Centrifuge cells for 30 sec in an E-tube.
- Discard the supernatant solution and resuspend the cells in 0.1 mL GTE solution (Solution I) containing Lyzozyme (4 mg/mL).
- Incubate for 10 min at RT (room temperature).
- Add 0.2 mL of freshly prepared solution (1 % SDS and 0.2 N NaOH) and shake to mix.
- Incubate for 1 min at RT.
- Add 30 mL Phenol equilibrated with two volume of SDS/NaOH solution. Vortex gently for a few seconds.
- Add 150 mL of 3 M NaOAc (pH 4.8) and shake the tube briefly.
- Incubate the tube at -20 ° C for 20 min.
- Centrifuge for 5 min. Tranfser the supernatant solution into new tube.
- Fill the tube with ice-cold Abs. Et-OH.
- Incubate for 30 min ~ 1 hr.
- Centrifuge for 5 min.
- Decant the su-pernatant solution and add 1 mL of an ice-cold 70 % Et-OH.
- Centrifuge for 1 min.
- Discard all the supernatant solution. Dry briefly in a hood.
- Resuspend the pellet in 30 mL of TE.
