Polysaccharide and Polyphenol Plant Total RNA (Plus) Small Quantity 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 kit provides a fast and efficient method for purifying polysaccharide and polyphenol plant total RNA, suitable for most polysaccharide and polyphenol-rich plant tissues. In general, plant tissues rich in polysaccharides and polyphenols contain a high level of secondary phenolic metabolites and polysaccharides, significantly affecting RNA extraction efficiency. This kit can effectively remove polysaccharides and polyphenols, as well as efficiently eliminate DNA contamination from samples. If the experiment is sensitive to DNA, it is recommended to use primers spanning introns for downstream experiments.

The RNA Fast Purification Kit can extract total RNA from plant tissues (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. 整個純化過程不需要苯酚氯仿等有毒試劑, making this RNA purification kit suitable for various other sample types.

5. 實驗原理和程序

6. 提取過程

開始實驗前的注意事項:

A. 使用前 洗滌緩衝液 1, add the specified amount of absolute ethanol according to the label on the reagent bottle, 並勾選標籤上的方框，表示已添加無水乙醇.

乙. 洗脫緩衝液是 0.1x TE 解決方案 含有極少量的 EDTA. EDTA是否影響後續實驗, 建議用無菌去離子水更換洗脫緩衝液.

1. 樣品加工:

A. 材料收集和儲存:

如果新鮮材料不能立即使用, 將它們放入液態氮中並儲存在-80°C.

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 using liquid nitrogen.

3. 添加 600μl RNA Extraction Buffer I, ensuring there are no blocky tissues in the ground sample. Blocky tissues are difficult to lyse and can reduce RNA yield.

4. Vortex for 30 秒.

5. 將裂解液離心 5 分鐘在 12,000 轉速.

(筆記: Polysaccharide plant materials may produce a lot of sticky substances at this step, which can shear RNA in subsequent steps. 所以, remove these substances during this step.)

6. Transfer the supernatant obtained in the previous step to a DNA Clear Purification Column, 離心機在 12,000 轉速為 30 秒, 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. Add the liquid to the RNA purification column, 離心機在 12,000 轉速為 30 秒, and discard the flow-through.
8. 添加 700µl 抑制劑去除緩衝液 至RNA純化柱, 離心機在 12,000 轉速為 30 秒, and discard the flow-through.
9. 添加 700µl 洗滌緩衝液 1 至RNA純化柱, 離心機在 12,000 轉速為 30 秒, and discard the flow-through.
10. 添加 500µl 洗滌緩衝液 1 至RNA純化柱, 離心機在 12,000 轉速為 3 分分鐘, and 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: 確認洗滌緩衝液中添加了乙醇 1. The presence of ethanol significantly affects subsequent experiments. 所以, membrane drying is crucial. 離心後, ensure the absence of ethanol before elution. 丟棄廢棄物和收集管. 使用洗滌緩衝液後 1, RNA純化柱上的薄膜應該只有輕微的顏色. 離心後小心取出RNA純化管柱, 確保它不接觸收集管以避免乙醇污染.)

12. 滴 50-100µl 洗脫緩衝液 到膜上, 離心機在 12,000 轉速為 1 分分鐘, and collect the RNA solution.

(筆記: Eluting RNA with 50μl Elution Buffer can increase RNA concentration but decrease total RNA yield.)