Notes
- This protocol describes a method for preparing Agrobacterium for freeze-thaw transformation 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 stock culture – from a glycerol stock.
- DNA for transformation – for test of competency.
- Use 5 µl of standard E. coli mini-prep DNA or 1-5 µg of CsCl-purified DNA.
- LB liquid medium.
- LB Agar plates plus appropriate antibiotics.
- 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.
- 1X TE buffer.
Equipment
- Incubator, preset to 28°C
- Rocking platform
- Autoclaved 500 mL flask.
- Sterile Tubes, 15-ml
- Sterile Tubes, micro-centrifuge (1.5 mL)
- Water bath, preset to 37°C
Method
Preparation of Competent Cells
- Prepare a 2.5 mL overnight culture of the chosen strain of Agrobacterium. Often best to prepare streak plate from glycerol stocks on LBA plates with appropriate antibiotics to check for contamination.
- Inoculate 200 ml of LB in a 500 mL flask with 1 ml of overnight culture with appropriate antibiotics. Incubate at 28°C with vigorous agitation.
- Grow the cells to log phase (OD550 0.5-0.8). Often takes 24 hrs.
- Pellet the cells in a bench-top centrifuge at 4000 rpm for 15 minutes at 4°C. Agrobacterium takes longer to pellet than E. coli.
- Wash the pellet with sterile 1X TE.
- Re-suspend the cells in 0.1X the original volume of LB, and aliquot 250µl fractions in micro-centrifuge tubes.
- Snap-freeze in liquid nitrogen and store at -70°C.
- Check viability by plating onto LBA plates with appropriate antibiotics and competency by transforming with freeze-thaw method with a control plasmid. Also, consider doing a negative control. Agro stocks can be contaminated by transformed strains.
Antibiotics for Agrobacterium strain selection
| Agrobacterium strain | Chromosomal marker | Ti plasmid marker |
|---|---|---|
| LBA4404 | Rifampicin | Spectinomycin & Streptomycin |
| GV2260 | Rifampicin | Carbenicillin |
| GV3101::pMP90 | Rifampicin | Gentamycin |
| GV3101::pMP90RK | Rifampicin | Gentamycin and Kanamycin |
| AGL-1 | Rifampicin | Carbenicillin |
| General Agro. selection marker | tetracycline |
- For GV3101 use 5 µg/ml tetracycline + 50 µg/ml rifampicin + 50 µg/ml kanamycin + 25 µg/ml gentamycin
