Freeze-Thaw Transformation of Agrobacterium tumafaciens

Notes

• This protocol describes a method for transforming Agrobacterium with plasmid DNA using a freeze-thaw technique. Although the transformation efficiency for Agrobacterium is lower than that for E. coli, it is possible to obtain adequate numbers of transformants with this technique.

Source

• This protocol was adapted from “How to Transform Arabidopsis,” Chapter 5, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

Materials

Reagents

• Agrobacterium competent cells.
• DNA for transformation.
  • Use 5 µl of standard E. coli mini-prep DNA or 1-5 µg of CsCl-purified DNA.
• LB liquid medium.
• LB liquid medium and agar plates containing appropriate antibiotics.
• Liquid nitrogen.
  • Caution: Liquid nitrogen (LN₂) can cause severe damage due to its extreme temperature. Handle frozen samples with extreme caution. Do not breathe the vapours. Liquid nitrogen in frozen vials that are immersed in liquid nitrogen can cause the vials to explode when they are removed. Wear gloves and eye goggles. Do not allow the liquid nitrogen to spill onto clothing. Do not breathe the vapours. Users should be trained and have read the risk assessment and safe operating procedure before using liquid nitrogen for the first time.

Equipment

• Incubator, preset to 28°C
• Rocking platform
• Sterile Tubes, micro-centrifuge (1.5 mL)
• Water bath, preset to 37°C

Method

Transformation and Recovery

1. Thaw competent Agrobacterium on ice (use 250 µl per transformation reaction), and add DNA (10 µl of standard E. coli mini-prep DNA or 1-5 µg of CsCl-purified DNA).
2. Keep the mixture on ice for 5 minutes
3. Wearing eye protection and gloves, transfer to liquid nitrogen for 5 minutes. 
4. Wearing eye protection and gloves and using forceps, remove the tubes from liquid nitrogen and incubate the mixture for an additional 5 minutes in a 37ºC water bath.
5. 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.
6. Collect the cells by spinning briefly in a micro-centrifuge, and spread them on one LB agar plates containing the appropriate antibiotic. Include the appropriate antibiotic for the T-DNA vector.
7. Incubate the cells for 2 days at 28°C.

Antibiotics for Agrobacterium strain selection

• For GV3101 use 5 µg/ml tetracycline + 50 µg/ml rifampicin + 50 µg/ml kanamycin + 25 µg/ml gentamycin