Rice Bombardment

Thursday, December 04, 2003

Description
Rice Bombardment

Procedure
DAY 1

1. Filtering Rice cells with 640 兪m mesh to get small size cells.

2. Discarding the sup

3. Mountaining #4 whatman paper on the N6D 0.5 media

4. Spread 250mg cell on the paper circulary, 1-layerd.

5. Store 4 hours, in the Dark

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6. Add M10 10兪L to 20兪L DNA soln. Mixing

7. Add CaCl2 25兪L to it, Vortexing 10 sec. (Aggregation)

8. Add Spermidine 10兪L to it, Tapping, Vortexing 10 sec. (Store in Ice, 5 min)

9. Bombardment vacuum sterilising (pressure > 65)

10. Preparation Bombardment appratus

He gas: Red valve, Open
Blue valve, Close (60 psi: optimum for Rice)
Green valve, Open

11. Spin Down DNA soln.

12. Discarding 50兪L sup. Use the rest, 15兪L for Bombardment (Twice shootable)

13. Tapping the rest DNA soln. with the pipette

14. Mountain 5兪L DNA soln. on a small filtering kit

15. Assemble the kit, place a Rice cell petridish 6 th location from the bottom

16. Vacuum the apparaus by pressure > 65, and SHOOT!

17. Store the Rice cell in the Dark for 24 hours

DAY 2

18. Transfer them to N6D (No osmotic)

DAY 3

19. After 24 hours, Analysis only one (UV irradiatoin)

20. After 2 - 3 days, Transfer them to N6D G418 (75~100mg/L) or Hygromycin B (50mg/L)

21 After 2 weeks, Transfer them to New N6D G418 or HygB

22. Select Antibotic resistant calluses in 6-8 weeks

Recipes
Apparatus

Small filtering kit 5 ea
Baffle (200兪m-400兪m stainless mesh)
#4 whatman paper
620兪m mesh in 儮flask
Yellow tips
N6D 0.5 solidified media

N6 (pH 5.6-5.8)
2,4-D 2 mg/L
Sorbitol 0.25 M (45.6 g/L)
Mannitol 0.25 M (45.6 g/L)
agar 8 g/L (phytagel 2 g/L)
N6D

N6 (ph 5.6-5.8)
2,4D- 2 mg/L
agar 8 g/L (phytagel 2 g/L)
N6D G418

N6D
G418 100 mg/L
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Filtering Kit 2 ea (one for CaCl2, the other for Spermidine)

Blue tips

Epp tubes

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Supplies

Tips

(责任编辑：泉水)

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Rice Bombardment