Introducing Binary plasmid into Agrobacterium by electroporation

INTRODUCING BINARY PLASMID INTO AGROBACTERIUM BY ELECTROPORATION

Last Update: December 2006

PROCEDURE

1. Remove from -80^oC tubes containing 80 mL of electro-competent Agrobacterium tumefaciens strain LBA4404, or GV3100 cells

2. Thaw the cells on ice

3. Add ~3 mg of Binary vector into the Agrobacterium cells. Incubate on ice up to 3 minutes

4. Transfer the mixture of cells+DNA to a cold electroporation cuvette (0.2 cm electrode gap). Make sure the suspension is at the bottom of the cuvette

5. Set the Gene Pulser apparatus (Bio-Rad) at 25 mF the volt at 2.5 kV. Set the Pulse resistance controller to 200 ohms

6. Place the cold cuvette in the chamber slide (Cuvette notch facing away from you)

7. Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber

8. Electroporate by pushing both red bottoms, until a beep sound (~4.6-4.8 seconds)

9. Remove the cuvette from the chamber and immediately add 1 mL of YEP/LB medium to the cuvette and quickly resuspend the cells by pipetting

10. Transfer the cell suspension from the cuvette to 1.5 mL tubes and incubate on shaker (200 rpm) at 28^oC for 2 h to allow recovery and expression of the antibiotic resistance markers (Clean cuvettes successively with dH₂O, EtOH, sterile water, then wrap with aluminum foil then autoclave)

11. Pipette 200 mL of each transformation on YEP plates containing selection

For PGA use YEP/Kan (12.5 mg/mL)/ tetracycline (5 mg/mL) - in Agro strain LB44040 add streptomycin (50 mg/mL); In Agro strain GV3100 add 50 mg/mL gentamycin

12. Spread the cells with bent glass rod. Place plates inverted at 28^oC for 2-3 days in the dark

13. Check for proper transformation by doing a plasmid mini prep and checking for the insert through a restriction digest