Protocol

Transformation of Agrobacterium Using Electroporation

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 electroporation in a manner similar to that commonly used for Escherichia coli. Although the transformation efficiency for Agrobacterium is lower than that for E. coli, it is possible to obtain adequate numbers of Agrobacterium transformants with this technique.

RELATED INFORMATION

An alternative approach for Agrobacterium transformation is the freeze-thaw method (see Transformation of Agrobacterium Using the Freeze-Thaw Method). Electroporation is more efficient because it requires less DNA, but an advantage to the freeze-thaw method is that it involves no 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 1 µl of an E. coli miniprep (see, e.g., Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation) or 1-5 µg of CsCl-purified plasmid DNA. DNA for electroporation must be free of salt, RNA, and protein. DNA in 1X TE buffer should be precipitated with ethanol (see, e.g., Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes) and resuspended in H₂O.

Glycerol (10% [v/v]), sterile, ice-cold

LB liquid medium (without antibiotics, see Steps 1 and 11)

LB liquid medium and agar plates containing appropriate antibiotics (see Step 12)

Sterile H₂O, ice-cold

Liquid nitrogen

Equipment

Cuvettes for electroporation, chilled

Centrifuge with rotor prechilled to 4°C

Electroporator (e.g., Bio-Rad)

Incubator, preset to 28°C

Parafilm

Pipettes, wide-bore

Spectrophotometer

Tubes, 15-ml

Tubes, microcentrifuge

Water bath, ice-cold

METHOD

This protocol was adapted from Shen and Forde (1989) and Mersereau et al. (1990).

Preparation of Competent Cells

1. Inoculate 500 ml of LB (not YEP) with 5 ml of a fresh saturated culture of the appropriate strain of Agrobacterium. Incubate the culture at 28ºC with vigorous agitation. Start the culture in the late afternoon, and harvest it the following morning.

2. When the cells have reached log phase (OD₅₅₀ 0.5-0.8), chill the culture by gently swirling it in an ice-water bath. Keep the cells at 4ºC for all further steps.

3. Pellet the cells by centrifuging at 4000g for 10 minutes at 4ºC in a prechilled rotor.

4. Discard the supernatant, add 5-10 ml of ice-cold H₂O, and use a wide-bore pipette to pipette the cells gently up and down until no clumps remain. Adjust the suspension volume to 500 ml with ice-cold H₂O.

5. Repeat Steps 3 and 4 twice.

i. After the first repeat, resuspend the cells in a final volume of 250 ml of ice-cold H₂O.

ii. After the second repeat, resuspend the cells in a final volume of 50 ml of ice-cold H₂O.

6. Pellet cells as in Step 3, and resuspend them in 5 ml of 10% (v/v) ice-cold, sterile glycerol.

7. Dispense 50-µl aliquots of cells into microcentrifuge tubes. Snap-freeze them in liquid nitrogen, and store them at -70ºC.

Electroporation and Recovery

8. Thaw competent cells on ice (50 µl per transformation).

9. Add plasmid DNA (1 µl of E. coli miniprep or 1-5 µg of CsCl-purified plasmid DNA) to the cells, and mix them together on ice.

10. Transfer the mixture to a prechilled electroporation cuvette. Carry out electroporation as recommended for E. coli by the manufacturer of the chosen electroporator.
For example, when using a Bio-Rad electroporator with a 2-mm cuvette, use the following conditions:

Capacitance: 25 µF

Voltage: 2.4 kV

Resistance: 200

Pulse length: 5 msec

11. Immediately after electroporation, add 1 ml of LB to the cuvette, and transfer the bacterial suspension to a 15-ml culture tube. Incubate for 4 hours at 28ºC with gentle agitation.

12. Collect the cells by centrifuging briefly, and spread them on an LB agar plate containing the appropriate antibiotic (see Table 1). Include the antibiotic for the T-DNA vector.

13. Incubate the cells for 3-4 days at 28ºC.

14. Restreak colonies on a new LB agar plate. Incubate this plate at 28ºC, and, when the colonies have grown, seal the plate with Parafilm. Keep it at 4ºC as a stock plate.

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

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

REFERENCES

Mersereau M., Pazour G.J., Das A. 1990. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90: 149–151.[Medline]

Shen W.J. and Forde B.G. 1989. Efficient transformation of Agrobacterium spp. by high-voltage electroporation. Nucleic Acids Res. 17: 8385.[Free Full Text]