石蠟包埋組織 DNA 萃取試劑盒

1. 試劑盒的組成

規格	50時間	100時間
貓. 不.	SN0251	SN0252
DNA萃取柱 (放)	50 (放)	100 (放)
試劑緩衝液IV	20 毫升	2 × 20 毫升
試劑緩衝液C	30 毫升	2 ×30毫升
Reagent Buffer FF	60 毫升	2 ×60毫升
洗滌緩衝液 1	15 毫升	2 × 15 毫升
洗脫緩衝液	20 毫升	20 毫升
蛋白酶K	1毫升	2x1毫升
rnasea	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℃) 並且在乾燥條件下, 保存期限為 12 月. DNA 萃取純化柱可保存 1 在涼爽乾燥的環境中度過一年. 蛋白酶 K 和 核糖核酸酶A 含防腐劑, 允許在室溫下運輸, 但為了長期儲存, 應保存在-20℃.

3. 試劑盒使用說明

3.1 本試劑盒適用於分子生物學研究，不得用於疾病診斷或治療.

3.2 試劑盒中部分成分含有刺激物. 建議採取防護措施，例如穿著防護衣和護目鏡.

3.3 本試劑盒使用過程中, 高速離心機, 水浴 (金屬浴), 渦旋混合器, 無水乙醇, 無菌去離子水, 和EP管需用戶自備.

4. 試劑盒簡介

 

Paraffin-embedded tissue DNA純化 kit provides a rapid and effective DNA purification solution for samples in paraffin sections. Tissues are collected after paraffin dissolution using a dedicated dewaxing solution, and sample DNA is obtained through subsequent extraction processes.

 

This kit can extract sample DNA from paraffin sections within 30 分分鐘, and the entire purification process does not require toxic reagents such as phenol-chloroform. 萃取的DNA可直接用於 聚合酶鍊式反應, 南方印跡, 和其他應用.

5. 實驗原理和程序

6. 提取過程

開始實驗前:

A. Reagent Buffer FF:This buffer should be stored long-term in an environment between 2°C and 8°C.

乙. Reagent Buffers IV and C 低溫條件下可能會沉澱. 建議加熱至65°C 5 分分鐘. 沉澱溶解後, the solution can be used normally.

C. 洗緩衝 1 should have a specified amount of anhydrous ethanol added before use, 如試劑瓶標籤上所示. Check the label with a tick mark to indicate the addition of anhydrous ethanol.

D. 洗脫緩衝液是 0.1x TE 解決方案 with a minimal amount of EDTA. EDTA是否影響後續實驗, it is advisable to replace the elution buffer with sterile deionized water.

 

1. 樣品加工:

Select 5-10 paraffin sections (1mm-3mm thickness), use scissors to remove excess wax, and cut the embedded tissue sample into small pieces. Place them in a 1.5ml centrifuge tube, 添加 800μl Reagent Buffer FF, incubate at 75°C for 5 minutes until all paraffin is dissolved. Centrifuge at 12000rpm for 5 分分鐘, 棄去上清液.

2. 添加 400μl Reagent Buffer IV, 20μL蛋白酶K (10 毫克/毫升), and 10μl RNaseA (10 毫克/毫升) to the precipitate from the previous step. Digest at 65°C, stirring every 6-7 直到消化完成的時間.
3. 加入等體積的 試劑緩衝液C和 an equal volume of anhydrous ethanol, 透過移液器充分混合.

(例如: If you add 400μl Reagent Buffer IV, approximately add 430μl Reagent Buffer C and 430μl anhydrous ethanol. Some precipitation may occur after adding Reagent Buffer C, but it does not affect subsequent experiments.)

4. 將所得液體加入DNA萃取純化管柱中 (or cartridge) (每次約650-700μl), let it stand at room temperature for 2 分分鐘, centrifuge at over 8,000rpm for 1 分分鐘. 將收集的廢物丟棄並將收集管重新插入下一步的淨化柱中.
5. 重複步驟 4, adding the remaining liquid to the DNA extraction purification column (or cartridge), centrifuge at over 8,000rpm for 1 分分鐘, 丟棄廢棄物和收集管.
6. 放置DNA萃取純化管柱 (or cartridge) 進入收集管, 添加 300微升 洗緩衝 1, centrifuge at over 8,000rpm for 1 分分鐘. Discard the waste and reinsert the purification column into the tube for the next step.

(筆記: Confirm that anhydrous ethanol has been added to 洗 緩衝 1.)

7. 添加 500微升 洗緩衝 1 至DNA萃取純化管柱 (or cartridge), centrifuge at 14,000rpm (20000×g) 為了 2 分分鐘. Extend centrifugation time if needed for a dryer membrane.
8. 放置DNA萃取純化管柱 (or cartridge) 在新的離心管中, 打開蓋子, 在65°C下孵育 2 分分鐘. Extend this step if necessary to evaporate ethanol to prevent residual ethanol from affecting downstream experiments.
9. Suspend the addition of 50-100µl 洗脫緩衝液 到膜, centrifuge at 12,000rpm for 2 分分鐘.

(筆記: 1. Using 50μl Elution Buffer for DNA elution can increase DNA concentration but decreases total DNA yield. 2. 洗脫的 DNA 可重新上樣至 DNA 萃取純化柱, centrifuged at 12000rpm for 2 minutes again to increase DNA yield.)