Complex Plant Total RNA (加) Small Amount Extraction Kit

1. 試劑盒的組成

2. 貯存

This reagent kit can be stored at room temperature (15-25℃) 在乾燥環境下穩定 12 月.

3. 試劑盒使用說明

3.1 該試劑盒旨在用於分子生物學研究目的，不應用於疾病診斷或治療.

3.2 套件中的某些成分含有刺激物; 建議採取必要的預防措施 (例如穿著防護衣和護目鏡).

3.3 使用該套件需要額外的設備，例如高速離心機, 水浴 (金屬浴), 渦旋混合器, 無水乙醇, 液態氮, 氯仿, 無菌去離子水, 和EP管.

4. 試劑盒簡介

This RNA purification reagent kit provides a fast and effective purification of plant total RNA containing polysaccharides, lipids, polyphenols, and other components. It is suitable for most complex plant tissues. In general, lipid-rich plant tissues contain a high content of lipid compounds, which significantly affect RNA extraction efficiency. This kit utilizes exclusive inhibitor removal columns to effectively eliminate lipids from plant samples, as well as to remove DNA contamination. If the experiment is sensitive to DNA, it is recommended to use intron-spanning primers for downstream experiments.

The RNA rapid purification reagent kit can extract plant total RNA (包括核RNA和細胞質RNA) 之內 1 小時. 萃取的RNA可直接用於RT-聚合酶鍊式反應, 北方印跡, ETC. The entire purification process does not require toxic reagents such as chloroform, making the RNA purification reagent kit suitable for various other samples.

5. 實驗原理和程序

6. 提取過程

開始實驗前的注意事項:

A. 洗 緩衝 1: Prior to use, add the specified amount of absolute ethanol as indicated on the reagent bottle label. Check the label to confirm the addition of absolute ethanol.

乙. 洗脫緩衝液是 0.1x TE 解決方案 with a minimal amount of EDTA. If EDTA may affect subsequent experiments, it is recommended to replace the elution buffer with sterile deionized water.

C. RNA Extraction Buffer I contains a small amount of phenol, which may precipitate. 使用前, mix thoroughly by warming in a water bath; after use, store away from light.

1. 樣品加工:

A. 材料收集和儲存:

如果新收集的材料無法立即使用, place them in liquid nitrogen and store them at -80℃.

乙. Whenever possible, collect fresh materials as they contain fewer polysaccharides and polyphenols.

2. Grind approximately 100 mg of fresh samples or not more than 20 mg of dry material with liquid nitrogen.

3. 添加 600μl of RNA Extraction Buffer I, ensuring there are no tissue clumps in the ground sample. Tissue clumps are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 s.

5. Transfer the lysate to aninhibitor removal purification column, 離心機在 12,000 轉速為 5 分分鐘.

(筆記: Lipid-rich plant materials may contain many lipid compounds at this step, which can affect RNA extraction. Remove these substances during this step. If buffer residue remains in the inhibitor removal purification column during centrifugation, extend centrifugation time appropriately.)

6. Transfer the obtained supernatant to a DNA removal column, 離心機在 12,000 rpm for 30s, and collect the filtrate (筆記: RNA is present in the filtrate).
7. 添加 250μl 無水乙醇, mix by pipetting. If there is a small amount of precipitation, it does not affect subsequent experiments. Transfer the liquid to an RNA purification column, 離心機在 12,000 rpm for 30s, discard the flow-through.
8. 添加 700μl of inhibitor removal buffer, 離心機在 12,000 rpm for 30s, discard the flow-through.
9. 添加 700微升的 washbuffer 1 至RNA純化柱, 離心機在 12,000 rpm for 30s, discard the flow-through.
10. 添加 500微升的 wash buffer 1 至RNA純化柱, 離心機在 12,000 轉速為 3 分分鐘, discard the flow-through.
11. Place the RNA purification column into a new 1.5ml centrifuge tube, air-dry the membrane at room temperature for 2 分分鐘.

(筆記: Confirm that absolute ethanol has been added to wash buffer 1. The presence of ethanol has a serious impact on subsequent experiments, so drying the membrane is crucial. 離心後, ensure no ethanol is present before elution, then discard the waste and collection tube. After washing with wash buffer 1, the membrane on the RNA purification column should have only a slight color. 離心後, carefully remove the RNA purification column, ensuring it does not touch the collection tube to avoid ethanol interference.)

12. Aerially pipette 50-100μl of elution buffer 到膜上, 離心機在 12,000 轉速為 1 分分鐘, and collect the RNA solution.

(筆記: 洗脫 RNA 50 μl of elution buffer can increase RNA concentration but reduces total RNA yield.)