Introduction of plasmid into Agrobacterium

Monday, November 10, 2003

Description
Introduction of plasmid into Agrobacterium

Procedure
Grow 10 ml ON culture at 28C in LB (+25mg/ml gentamycin for GV3101, + 34mg/ml rifampicin for LBA4404)
3. Dilute about 1:100 into 10mL of LB (with appropiate selection) and grow around 6-8 hours at 28C.
Note: Grow LBA4404 for a longer time since it grows slower.
4. Once the culture should reach a moderately dowdy stage (still in log-phase), take ~2 mL of the culture per transformation and transfer to a 2 mL eppendorf tube. Chill on ice for 1-5 minutes and then pellet for about 2-3 minutes in a microcentrifuge.
Note: checking ODs at this stage is not very useful since Agrobacterium cultures tend to accumulate dead cells.
5. Aspirate off the media and replace with 1 mL cold 10 mM Tris pH 7.5. Resuspend cells gently but completely. Pellet cells as above.
6. Resuspend cells in 200 ml of cold LB. Cells should resuspend well at this step.
Note: If the cells resuspend as clumps, you are better off starting over; they will not transform.
7. Add about 1-2 mg of plasmid DNA to the cells and freeze in liquid nitrogen for 5 minutes.
8. Transfer the cells quickly to a 37C water bath and incubate for 5 minutes.
9. Add 1ml of fresh LB and recover the cells at 28C for 2-3 hours.
10. Plate about 200-500 ml of the cells onto selection plates. Grow at 28C for ~2 days (for GV3101) to 3 days (LBA4404).

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Introduction of plasmid into Agrobacterium