Tissue DNA/RNA Extraction Kit

1. 試劑盒的組成

規格	50時間	100時間
貓. 不.	SN0329	SN0330
DNA Clean-Up Columns (放)	50 (放)	100 (放)
RNA萃取柱 (放)	50 (放)	100 (放)
RNA Extraction Buffer I	30 毫升	2×30 毫升
RNA Extraction Buffer IV	30 毫升	2×30 毫升
抑制劑去除緩衝液	2×30 毫升	4×30 毫升
洗滌緩衝液 1	2×15 毫升	3×15 毫升
洗脫緩衝液	20 毫升	20 毫升
蛋白酶K	1毫升	2x1毫升
使用說明書	1	1

 

2. 貯存

該試劑盒應在室溫下保存 (15-25℃) in a dry condition and is stable for 12 月. Proteinase K contains a stabilizer, allowing it to be transported at room temperature; 然而, for long-term storage, 應保存在-20℃.

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

The sample DNA/RNA simultaneous isolation and purification kit provide a fast and efficient purification solution for tissue sample DNA/RNA. This kit offers two sets of lysis systems, suitable for various sample tissues and cells, including most plants, 動物, 細菌, ETC.

The DNA/RNA rapid purification kit can extract total DNA/RNA from samples within 1 小時. The extracted DNA/RNA can be directly used for 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.

5. 實驗原理和程序

6. 提取過程

開始實驗前的注意事項:

A. 使用之前, 加入規定量的無水乙醇 洗緩衝 1 根據試劑瓶上的標籤, and mark a check on the label to indicate 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 has an impact on subsequent experiments, it is recommended to use sterile deionized water as a substitute for the elution buffer.

C. RNA Extraction Buffer I is designed for the extraction of plant and fungal samples, while RNA Extraction Buffer IV is primarily used for animal tissues, 血, and other sample tissues.

1. 樣品加工:
2. Plant/animal tissue materials: Collect samples using liquid nitrogen, grind the collected material directly in liquid nitrogen, and proceed to step 2 (recommended to use RNA Extraction Buffer I).
3. Cell tissues: Centrifuge and collect <107 suspended cells in a 1.5 毫升離心管, quickly proceed to step 2 (recommended to use RNA Extraction Buffer III).
4. 添加 500μl RNA Extraction Buffer I/RNA Extraction Buffer IV, 10μL蛋白酶K, vortex and mix thoroughly. Ensure gentle mixing in this step to prevent mechanical cutting of tissue DNA and reduce DNA yield.
5. Transfer the lysate to a DNA純化 柱子, 離心機在 13,000 轉速為 2 分分鐘, collect the filtrate (RNA is present in the filtrate). 在此刻, DNA is adsorbed on the DNA column and can be briefly stored at 4°C while simultaneously collecting RNA in the next steps.

(筆記: If the sample is plant tissue, 離心機在 13,000 轉速為 10 分分鐘, and add the supernatant to the DNA binding column.)

4. Precisely estimate the volume of the filtrate, 添加 5 times thevolume of absolute ethanol, 充分混合. 如果發生降水, it does not affect subsequent experiments.
5. Add the obtained liquid to the RNA extraction purification column (每次約650-700μl), 離心機超過 8,000 轉速為 1 分分鐘, 丟棄收集的廢液, 並將收集管重新插入純化管柱進行下一步.
6. 重複步驟 5, add the remaining liquid to the RNA extraction purification column, 離心機超過 8,000 轉速為 1 分分鐘, discard the waste liquid and collection tube, place the RNA extraction purification column in a new collection tube, and prepare for simultaneous collection of DNA.
7. 添加 600µl 抑制劑去除緩衝液 to the DNA extraction purification column and RNA extraction purification column, 離心機超過 8,000 轉速為 1 分分鐘, 丟棄廢液, and reinsert the DNA/RNA purification column into the collection tube for the next step.
8. 添加 700µl 洗滌緩衝液 1 to the DNA extraction purification column and RNA extraction purification column, 離心機在 14,000 轉速 (20,000×g) 為了 2 分分鐘. Extend centrifugation time appropriately to ensure the membrane is thoroughly dried.

(筆記: 確認洗滌緩衝液中已添加無水乙醇 1. The presence of ethanol has a significant impact on subsequent experiments, 因此膜乾燥至關重要. 離心後, 確保在洗脫前不存在乙醇, then discard the waste liquid and collection tube.

After washing with Wash Buffer 1, the membrane of the RNA purification column should only have a slight color. 離心後, carefully remove the RNA purification column, ensuring no contact with the collection tube to avoid ethanol interference.)

9. Place the DNA/RNA purification column into a new centrifuge tube, 滴 50-100 μl Elution Buffer onto the membrane, 室溫孵育 5 分分鐘 (15°C-25°C), 離心機超過 8,000 轉速為 1 分分鐘.

(筆記: Eluting nucleic acids with 50 μl Elution Buffer can increase the nucleic acid concentration but decrease the total nucleic acid yield.)

• 筆記:

1. 一般來說, larger elution volumes result in higher elution efficiency, but to increase nucleic acid concentration, the elution volume can be reduced, but not below 50 微升.
2. 核糖核酸, in the presence of RNA Extraction Buffer I/RNA Extraction Buffer III, is not degraded by RNAse but should be distinguished from DNA in subsequent experiments. Use RNAse-free consumables for RNA separation whenever possible.