CTAB法小規模植物基因組DNA萃取試劑盒

1. 試劑盒的組成

2. 貯存

該套件應在室溫下保存 (15-25℃) 乾燥條件下可保存 12 月. DNA萃取純化柱可在陰涼乾燥的環境中保存長達 1 年. RNase A 含有防腐劑，可在室溫下運輸, 但為了長期儲存, 應保存在-20℃.

3. 試劑盒使用說明

3.1 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

這 CTAB-based Plant DNA純化 Kit provides an improved CTAB method for purifying DNA, utilizing a specific binding buffer that efficiently precipitates DNA, and subsequently collects high-purity DNA through an adsorption column.

This kit is widely used for plant tissues and fungi, capable of extracting total DNA from samples within 2 小時 (including mitochondrial DNA and chloroplast DNA). 萃取的DNA可直接用於下游實驗，例如 聚合酶鍊式反應, 南方印跡, 和別的.

5. 實驗原理和程序

6. 提取過程

開始實驗前的注意事項:

1. Reagent buffers A and C 低溫條件下可能會沉澱. It is recommended to heat at 65°C for 5 minutes and use after the precipitates have dissolved.
2. 洗緩衝 1 should have the specified amount of anhydrous ethanol added as indicated on the bottle label. Mark the label once ethanol has been added.
3. Elution buffer is a 0.1x TE 解決方案含有極少量的 EDTA. EDTA是否影響後續實驗, sterile deionized water is recommended as a substitute for the elution buffer.

1. 樣品處理:
2. 材料收集和儲存:

Freshly collected material, if not immediately used, should be placed in liquid nitrogen and ultimately stored at -80°C. 乾燥後的物料可在室溫下保存.

1. If possible, collect fresh material as it contains fewer polysaccharides and polyphenols.
2. 從液體培養物中收集真菌時, separate the liquid by centrifugation and collect the fungal bodies.
3. Grind around 100 mg 新鮮樣品或不超過 20 mg of dry material using liquid nitrogen.

(筆記: Different sample quantities may require optimization through preliminary experiments before use.)

3. 添加 550 μl of reagent buffer A and 10 µl RNase A (10 毫克/毫升) to ensure there are no tissue clumps in the ground sample. Tissue clumps are difficult to lyse and can reduce DNA yield. Do not mix reagent buffer A and RNase A使用前.
4. 65°C 下孵育 20-30 分分鐘, gently invert 2-3 次. This step is for cell lysis.
5. 將裂解液離心 5 分鐘在 14,000 轉速 (20,000×g).

(筆記: Some plant materials may have a lot of sticky substances at this step, which can shear DNA in subsequent steps. Ideally, remove these substances by transferring the supernatant to a new centrifuge tube after centrifugation.)

6. Carefully transfer the liquid obtained in the previous step to a new centrifuge tube.

(筆記: 大約 500 可轉移μl液體; 對於某些物種, it may be less than 500 微升.)

7. Add an equal volume of chloroform to the lysate and gently invert to mix.

(筆記: 例如, 添加 500 μl of chloroform if you have 500 µl 裂解液. 如果裂解液體積小於 500 微升, adjust the chloroform volume accordingly.)

8. 離心機在 12,000 轉速為 10 分分鐘.
9. 小心地將上清液轉移至新的離心管中 (大約 500 微升).
10. 加入等體積的 reagent buffer C and an equal volume of anhydrous ethanol to the lysate, 並混合.

(例如, if you add 450 μl of reagent buffer C, 然後加 450 μl of anhydrous ethanol. 如果裂解液體積小於 450 微升, reduce the amount of reagent buffer C proportionally. Some precipitation will occur upon adding reagent buffer C, but it will not affect subsequent experiments.)

11. Transfer the obtained liquid to a DNA purification column (成套工具), 大約 650-700 μl each time. Centrifuge at over 8,000 轉速為 1 分分鐘, 丟棄收集的廢棄物, 並將收集管重新插入純化管柱進行下一步.
12. 重複步驟 11, adding the remaining liquid to the DNA purification column (成套工具) and centrifuge at over 8,000 轉速為 1 分分鐘. 丟棄廢棄物和收集管.
13. Place the DNA purification column (成套工具) into a new collection tube, 添加 300 微升的 洗 緩衝 1, 離心機超過 8,000 轉速為 1 分分鐘, 丟棄廢棄物, and reinsert the DNA purification column (成套工具) 進入管中進行下一步.

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

14. 添加 500 μl of Rinse Buffer 1 to the DNA purification column (成套工具), 離心機在 14,000 轉速 (20,000×g) 為了 2 分分鐘, slightly prolong the centrifugation time for a drier membrane.
15. Place the DNA purification column (成套工具) 放入新的離心管中, open, and heat at 65°C for 2 分分鐘. This step may be prolonged to evaporate ethanol as much as possible to prevent residual ethanol from affecting downstream experiments.
16. 滴 100 μl of elution buffer到膜上, 離心機在 12,000 轉速為 2 分分鐘.

(筆記: 1. Eluting DNA with 50 μl elution buffer can increase DNA concentration but decrease total DNA yield. 2. The eluate can be reapplied to the DNA purification column for a second elution, 離心機在 12,000 轉速為 2 minutes to collect, which may improve DNA yield.)