AGROBACTERIUM VIR GENE INDUCTION PROTOCOL

Thursday, December 04, 2003

Description
AGROBACTERIUM VIR GENE INDUCTION PROTOCOL

Procedure
1. Grow Agrobacterium at 30oC overnight in 5 ml rich medium (such as LB or YEP) containing the appropriate antibiotics.

2. Dilute approximately 0.5 ml of the culture into 50 ml AB-sucrose minimal medium containing the appropriate antibiotics. Grow overnight at 30oC until the bacteria are in late-log phase (Klett=100 on a Klett-Somerson Spectrophotometer, red filter).

3. Spin down the bacteria. Resuspend in two volumes of induction medium containing 100 uM acetosyringone. Shake (very gently) 14-24 hours at room temperature (not at 30oC).

4. Spin down the bacteria. Resuspend in MS plant tissue culture medium. Inoculate plants.

5. After two days, rinse the plant tissue in medium containing 100 ug/ml timentin. Continue incubating the tissue on solidified medium containing timentin.

6. Stain the tissues after various periods of time (2-10 days) in X-gluc.

Recipes
AB Medium:

20x AB Buffer:

K2HPO4 60 g/l
NaH2PO4 20 g/l

Autoclave separately

20x AB Salts:

NH4Cl 20 g/l
MgSO4.7H2O 6 g/l

KCl 3 g/l

CaCl2 0.2 g/l

FeSO4.7H2O 50 mg/l

pH to 7 before autoclaving

Combine 50 ml AB Buffer and 50 ml AB Salts with 900 ml sucrose-water (final concentration of sucrose in one liter is 0.5%).

Induction medium:

1x AB salts
2 mM NaPO4

50 mM MES, pH 5.6

0.5% glucose

100 uM acetosyringone

Supplies

Tips

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AGROBACTERIUM VIR GENE INDUCTION PROTOCOL