rif rif rif, carb gent gent and kan

13. Incubate the cells for 3-4 days at 28°C.

14. Restreak colonies on a new LB agar plate. Incubate this plate at 28°C and, when colonies have grown, seal with Parafilm and keep at 4°C as a stock plate.

15. Grow small liquid cultures of the restreaked colonies and carry out minipreps and/or polymerase chain reaction (PCR) to verify the presence of plasmid DNA (see PCR Analysis of Agrobacterium below).

16. Make glycerol stocks of the appropriate clones and store them at

Transformation of Agrobacterium Using the Freeze-Thaw Method

This protocol was adapted from Hofgen and Willmitzer (1988). Materials

Stock culture of the appropriate Agrobacterium strain DNA for transformation Liquid nitrogen

CAUTION: liquid nitrogen (see Appendix 3) Procedure

Preparation of Competent Cells

1. Inoculate 200 ml of LB with 1 ml of an overnight culture of the chosen strain of Agrobacterium. Incubate at 28°C with vigorous agita

tion. Start the culture in the late afternoon, to harvest the following morning.

2. Grow the cells to log phase, i.e., OD55Q 0.5-0.8.

3. Pellet the cells in a bench-top centrifuge at 5000 rpm for 10 minutes at room temperature.

Agrobacterium takes longer to pellet than E. coli.

4. Wash the pellet with sterile TE.

5. Resuspend the cells in 1/10 the original volume of LB and aliquot 250- or 500-jll fractions in microcentrifuge tubes.

6. Snap freeze in liquid nitrogen and store at -70°C.

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