Biology Reference
In-Depth Information
2
Materials
Prepare all solutions using ultrapure water and analytical grade
reagents. Store all the solutions as indicated. When disposing waste
materials, follow waste disposal regulations.
2.1 Plant Grains
and Buffers for OB
Isolation
Prepare fresh solutions for each isolation. Keep all the buffers
at 4 °C.
1. The grains of
Arabidopsis thaliana
, ecotype Columbia, should
be stored in a dark, cold (4 °C), and dry place (
see
Note 1
).
2. Phosphate stock solution: 80 mM sodium phosphate, pH 7.5.
Weigh 1.25 g of monosodium phosphate dihydrate into a glass
beaker and dissolve in 80 mL of water. Mix and adjust pH with
NaOH (
see
Note 2
). Make up to 100 mL with water in a grad-
uated cylinder.
3. Isolation buffer 1 (IB1): 0.6 M sucrose, 10 mM sodium phos-
phate, pH 7.5. Weigh 16.43 g of sucrose, add 10 mL of
phosphate stock solution, and make up to 80 mL with water.
4. Isolation buffer 2 (IB2): 0.4 M sucrose, 10 mM sodium phos-
phate, pH 7.5. Weigh 10.95 g of sucrose, add 10 mL of
phosphate stock solution, and make up to 80 mL with water.
5. Isolation buffer 3 (IB3): 0.2 M sucrose, 0.1 % Tween 20 (v/v),
5 mM sodium phosphate, pH 7.5. Weigh 2.74 g of sucrose,
add 2.5 mL of phosphate stock solution and 400
L of 10 %
Tween 20 (v/v,
see
Note 3
). Make up to 40 mL with water.
6. Isolation buffer 4 (IB4): 10 mM sodium phosphate, pH 7.5.
Dilute 10 mL of phosphate stock solution up to 80 mL with
water.
7. Isolation buffer 5 (IB5): 0.6 M sucrose, 2 M sodium chloride,
10 mM sodium phosphate, pH 7.5. Weigh 8.22 g of sucrose
and 4.68 g of sodium chloride, add 5 mL of phosphate stock
solution, and make up to 40 mL with water.
8. Isolation buffer 6 (IB6): 0.25 M sucrose, 2 M sodium chlo-
ride, 10 mM sodium phosphate, pH 7.5. Weigh 3.42 g of
sucrose and 4.68 g of sodium chloride, add 5 mL of phosphate
stock solution, and make up to 40 mL with water.
9. Urea: 9 M solution in water. Weigh 10.81 g of urea and dis-
solve in water to reach the fi nal volume of 20 mL (
see
Note 4
).
10. Hexane.
11. Storage buffer: 50 mM phosphate buffer, pH 7.5. Mix 5 mL
of phosphate stock solution and 3 mL of water.
μ
2.2 Solutions
for SDS-PAGE
1. Resolving gel buffer: 1.5 M Tris-HCl, pH 8.8. Weigh 18.2 g
of Tris-HCl into a glass beaker and dissolve in about 80 mL of
