Protocol

Transformation of Agrobacterium Using the Freeze-Thaw Method

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.

INTRODUCTION

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 Escherichia coli, it is possible to obtain adequate numbers of transformants with this technique.

RELATED INFORMATION

Agrobacterium may also be transformed using electroporation (see Transformation of Agrobacterium Using Electroporation Although electroporation requires less DNA and therefore is more efficient, an advantage of the freeze-thaw method is that it does not require any special equipment.

To generate transgenic Arabidopsis thaliana using Agrobacterium-mediated transfer, see In Planta Transformation of Arabidopsis.

MATERIALS

Reagents

Agrobacterium stock culture

See Vectors and Agrobacterium Hosts for Arabidopsis Transformation for considerations regarding Agrobacterium strains and T-DNA vectors.

DNA for transformation

Use 10 µl of standard E. coli miniprep DNA (see, e.g., Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation) or 1-5 µg of CsCl-purified DNA.

LB liquid medium

LB liquid medium and agar plates containing appropriate antibiotics (see Step 11)

Liquid nitrogen

TE buffer (10X)

Equipment

Incubator, preset to 28°C

Rocking platform

Tubes, 15-ml

Tubes, microcentrifuge

Water bath, preset to 37°C

METHOD

This protocol was adapted from Höfgen and Willmitzer (1988).

Preparation of Competent Cells

1. Inoculate 200 ml of LB with 1 ml of an overnight culture of the chosen strain of Agrobacterium. Incubate at 28ºC with vigorous agitation. Start the culture in the late afternoon to be harvested the following morning.

2. Grow the cells to log phase (OD₅₅₀ 0.5-0.8).

3. Pellet the cells in a benchtop centrifuge at 5000 rpm for 10 minutes at room temperature.
Agrobacterium takes longer to pellet than E. coli.

4. Wash the pellet with sterile 1X TE.

5. Resuspend the cells in 0.1X the original volume of LB, and aliquot 250- or 500-µl fractions in microcentrifuge tubes.

6. Snap-freeze in liquid nitrogen and store at -70ºC.

Transformation and Recovery

7. Thaw competent Agrobacterium on ice (use 250 µl per transformation reaction), and add DNA (10 µl of standard E. coli miniprep DNA or 1-5 µg of CsCl-purified DNA).

8. Keep the mixture on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes.

9. Incubate the mixture for an additional 5 minutes in a 37ºC water bath.

10. 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.

11. Collect the cells by spinning briefly in a microcentrifuge, and spread them on two LB agar plates containing the appropriate antibiotic (see Table 1). Include the antibiotic for the T-DNA vector.

12. Incubate the cells for 2 days at 28ºC.

13. Restreak colonies on a new plate, and incubate this plate for 2 days at 28ºC. Seal the plate with Parafilm and keep it as a stock plate.

14. Grow small liquid cultures of the restreaked colonies, and carry out minipreps and/or PCR to verify the presence of the plasmid DNA (see PCR Analysis of Agrobacterium.

15. Make glycerol stocks of the appropriate clones, and store them at -20ºC.

REFERENCES

Höfgen, R. and Willmitzer, L. 1988. Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16: 9877.[Free Full Text]