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Description Agrobacterium Transformation Procedure 1. Seed culture in YEP/Streptomycin+ media, 28¡É, shaking for overnight 2. Add 3-4 mL of overnight cultured cell to 50 mL of fresh YEP/Streptomycin+ media, 4 hr shaking (OD600¡Ö0.5) 3. 4000 rpm, 5 min, 4¡É, obtaining cell pellet 4. Resuspend cell pellet in 10 mL of 0.15 M NaCl (prechilled soln.) 5. 4000 rpm, 5 min, 4¡É, obtaining cell pellet 6. Add l mL of ice-cold 20 mM CaCl2 (-----------> the preparation of C Cell) 7. Transfer 200 ¥ìL of the C cell soln. into the glass tube 8. Add 1¥ìg / 20¥ìL DNA to the tube 9. Incubate on ice for 30 min 10. Freeze the cells in liquid nitrogen, 1 min 15 sec 11. Thaw the cells in 37¡É for 5-6 min 12. Add 1 mL of fresh YEP media to the tube 13. Transfer the soln. into ependorf tube 14. Incubate at 28¡É for 2-4 hr 15. Centrifuge 5 min, 4¡É, obtaining the cell pellet 16. Add 200 ¥ìL of YEP media 17. Spread the cells onto YEP/Km+ solid media 18. Incubate at 28¡É for 2-3 days Recipes YEP liquid and solid media Peptone 10 g/L Yeast extract 10 g/L NaCl 5 g/L pH 7.5 Agar 15 g/L, Km 50 ¥ìg/mL: for Solid medium Preparations A. tumefaciens LBA4404 (PC2760) 0.5M NaCl (Diluted with autoclaved DDW to final conc. 0.15 M) 20 mM CaCl2 Filtering kits containing 0.22 ¥ìm membrane Glass tube Blue and Yellow tips Supplies Tips (责任编辑：泉水) |
Agrobacterium Transformation
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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