Introduction of plasmid into Agrobacterium (method III)

INTRODUCTION OF PLASMID INTO AGROBACTERIUM (METHOD III)
	Last Update: December 2006


PREPARE SOLUTIONS
1. AB Medium:	Mix 2.5 g of sucrose in 450 mL of dH₂O, 25 mL of 20X AB Buffer, and 25 mL of 20X AB Salts (Autoclave)
2. 20X AB Buffer:	Mix 78.6 g of K₂HPO₄-3H₂O or 60.0 g of K₂HPO₄, 23.0 g of NaH₂PO₄-H₂O, and add dH₂O to 1 L (Autoclave)
3. 20X AB Salts:	Mix 20.0 g of NH₄Cl, 6 g of MgSO₄-7H₂O or 2.9 g of MgSO₄, 3 g of KCl, 0.26 g of CaCl₂-2H₂O or 0.2 g of CaCl₂, and 50 mg of FeSO₄-7H₂O. pH 7.0. dH₂O to 1 L (Autoclave)

PROCEDURE
The idea of this protocol is to introduce the plasmid of interest into Agrobacteria with the help of a helper E. coli bacteria containing a vector that helps in the mobilization of the vector from the bacteria to the Agrobacteria. A reference describing the helper vector is: Ditta G., Stanfield S., Corbin D., and Helinski DR. (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. PNAS 77: 7347-7351. A reference describing the whole protocol can be found in the following reference: Figurski DH., and Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid functin provided in trans. PNAS 76: 1648-1652
1. Grow overnight the following cultures in a shaking incubator:
1.1 Helper bacteria with helper vector (Kan⁵⁰) in LB medium (at 37^oC)
1.2 Your clone in LB medium (at 37^oC)
1.3 Agrobacteria into which your vector is to be transferred (GV3101, Gent⁵⁰), in AB medium (at 28^oC)
2. Mix 100 uL of each of the three cultures into a single tube
3. Pellet cells and resuspend in 100 uL of LB medium
4. Plate in an LB plate. IMPORTANT: grow at 30^oC
5. Recover lawn of cells using 5 mL of AB medium
6. Dilute cell solution (about 1,000) and plate in an AB plate containing antibiotics selecting for the Agrobacteria and for your clone of interest. Grow at 28^oC
Note: only Agrobateria will be able to grow under these conditions