Introduction of plasmid into Agrobacterium (method II)

INTRODUCTION OF PLASMID INTO AGROBACTERIUM (METHOD II)

Last Update: December 2006

PREPARE SOLUTIONS

1. LB media (1 L):

Mix 10 g of Bacto-tryptone, 5 g of yeast extract, and 10 g of NaCl. pH to 7.5 with NaOH and add dH₂O to 1 L (Autoclave)

2. 1M Tris, pH 7.5 (1 L):

Mix 121.1 g of Tris base with 800 mL dH₂O. Add ~60 mL cHCl. pH is temperature dependent: ~0.03 pH units per 1^oC increase. Make sure it is at RT before making final pH adjustments. Add dH₂O to 1 L (Autoclave)

3. 10mM Tris, pH 7.5 (1 L):

Mix 100 mL of 1M Tris, pH 7.5 with 900 mL of dH₂O

PROCEDURE

Enough for 4 transformations:

1. Grow 10 mL ON culture at 28^oC in LB (+25 mg/mL gentamycin for GV3101, + 34mg/mL rifampicin for LBA4404)

2. Dilute about 1:100 into 10 mL of LB (with appropiate selection) and grow around 6-8 hours at 28^oC

NOTE: Grow LBA4404 for a longer time since it grows slower

3. Once the culture should reach a moderately clowdy 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

4. Aspirate off the media and replace with 1 mL cold 10mM Tris pH 7.5. Resuspend cells gently but completely. Pellet cells as above

5. 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

6. Add about 1-2 mg of plasmid DNA to the cells and freeze in liquid nitrogen for 5 minutes

7. Transfer the cells quickly to a 37^oC water bath and incubate for 5 minutes

8. Add ~1 mL of fresh LB and recover the cells at 28^oC for 2-3 hours

9. Plate about 200-500 mL of the cells onto selection plates. Grow at 28^oC for ~2 days (for GV3101) to 3 days (LBA4404)

NOTE: efficiency can vary a lot, so plate at least two different dilutions to make sure individual colonies will be present

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