内容发布更新时间 : 2024/11/6 3:38:09星期一 下面是文章的全部内容请认真阅读。

Extraction buffer

200 mM Tris–HCl pH 7.3, 20 mM EDTA, 50 mM Na2SO3, 1.5 M urea,

2% (w/v) 2-h-mercaptoethanol

All purification steps or incubations were performed either on ice or at 4 -C.

1. 50–100 g of leaf tissue was homogenized in a blender in four volumes (w/v) of extraction

buffer.These and subsequent steps were performed at 4 -C.

2. Triton X-100 (2% w/v) was added to the leaf slurry, stirred 20 min, filtered through two

layers of cheesecloth and centrifuged for 10 min at 7000 rpm

3. The resulting supernatant wasfiltered through two layers of Miracloth and centrifuged for 45

min at 55,000 rpm .

4. The resulting pellet was rinsed with water and resuspended in 1 M urea solubilized in TE (10

mM Tris, 1 mM EDTA, pH 7.5).The mixture was stirred for 1 h then centrifuged for 5 min at 10,000 rpm.

5. The supernatant was layered on a 5–40% sucrose density gradient of 1 M urea in TE and

centrifuged for 2 h at 28,000 rpm

6. The resulting light-scattering zone was removed with a syringe and an 18-g needle, mixed

with an equal volume of 1. M urea in TE, and centrifuged at 55,000 rpm for 45 min in a Beckman Ti 60 rotor.

7. The pellet was gently rinsed with water, resuspended in 4 ml TE to which 1.2 g CsSO4 was

added and the mixture was centrifuged for 20 min at 10,000 rpm.

8. The resulting supernatant was transferred to QuickSeal tubes and centrifuged at 40,000 rpm

for 16 h in a Beckman SW 60 rotor.

9. A light-scattering band was collected, mixed with an equal volume of TE and centrifuged for

45 min at 55,000 rpm in a Beckman Ti 60 rotor.

10. The final pellet was resuspended in TE with 1% (w/v) sodium dodecyl sulfate (SDS), phenol

extracted and ethanol-precipitated The pellet was suspended in distilled water, and the integrity of the RNA was verified by agarose gel electrophoresis.