Description
Rice Bombardment

Procedure
DAY 1

1. Filtering Rice cells with 640 ƒÊm 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

6. Add M10 10ƒÊL to 20ƒÊL DNA soln. Mixing

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

8. Add Spermidine 10ƒÊL 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 50ƒÊL sup. Use the rest, 15ƒÊL for Bombardment (Twice shootable)

13. Tapping the rest DNA soln. with the pipette

14. Mountain 5ƒÊL 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 (200ƒÊm-400ƒÊm stainless mesh)
#4 whatman paper
620ƒÊm mesh in ƒ¢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


Supplies


Tips