細菌總RNA (加) 提取套件

1. 試劑盒的組成

規格	50時間	100時間
貓. 不.	SN0321-D	SN0322-D
RNA萃取柱 (放)	50（套）	100（套）
DNA Clean-Up Columns (放)	50（套）	100（套）
RNA Extraction Buffer I	30毫升	2× 30 毫升
抑制劑去除緩衝液	30毫升	2×30 ml
洗滌緩衝液 1	15 毫升	2×15 ml
洗脫緩衝液	20毫升	2×20毫升
溶菌酶	5毫升	2x5ml
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 environment and can be preserved for 12 月. Lysozyme contains a preservative, 允許在室溫下運輸; 然而, for long-term storage, it should be stored at -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

This RNA purification kit provides a rapid and efficient purification solution for bacterial RNA, suitable for most bacterial tissues. The kit employs DNA removal technology to effectively eliminate genomic DNA, and the resulting RNA samples typically do not require on-column digestion, reducing the risk of RNA degradation.

The RNA Fast Purification Kit allows for the extraction of total bacterial 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 involve toxic reagents such as phenol-chloroform, making the RNA purification kit well-suited for various 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, 加入規定量的無水乙醇 洗滌緩衝液 1 如試劑瓶標籤上所示, and mark with a check to indicate the addition of absolute ethanol.

乙. 洗脫緩衝液是 0.1x TE 解決方案 含有極少量的 EDTA. If EDTA may impact subsequent experiments, 建議用無菌去離子水更換洗脫緩衝液.

1. 樣品加工:

A. Gram-positive bacteria: Centrifuge the bacterial suspension at 4℃, 12,000 轉速為 2 分分鐘, collect bacterial cells (1.53 毫升), 棄去上清液, 添加 100µl 溶菌酶 (10毫克/毫升), thoroughly mix the bacterial cells, 並在室溫下孵育 15-30 分分鐘.

乙. 革蘭氏陰性細菌: Centrifuge the bacterial suspension at 4℃, 12,000 轉速為 2 分分鐘, collect bacterial cells (1.53 毫升), 棄去上清液, 添加 10µl 溶菌酶 (10毫克/毫升), thoroughly mix the bacterial cells, 並在室溫下孵育 3-5 分分鐘.

2.添加 400μl RNA Extraction Buffer I, vortex to mix thoroughly.

3. Transfer the lysate to a DNA clearance purification column, 離心機在 13,000 轉速為 2 分分鐘, collect the filtrate (RNA is present in the filtrate).

4. Accurately estimate the volume of the filtrate, 添加 0.5 times the volume of absolute ethanol, 充分混合; if precipitation occurs, it does not affect subsequent experiments.

5. Add the obtained liquid to the RNA extraction purification column (每次約650~700μl), 離心機轉速超過 8,000 轉速為 1 分分鐘, 丟棄收集的廢液, and reinsert the collection tube into the RNA extraction purification column for the next step.

6. 重複步驟 5, add the remaining liquid to the RNA extraction purification column, 離心機轉速超過 8,000 轉速為 1 分分鐘, discard the waste liquid and the collection tube.

7. Place the RNA extraction purification column into a new collection tube, 添加 600µl 抑制劑去除緩衝液, 離心機轉速超過 8,000 轉速為 1 分分鐘, 丟棄廢液, and reinsert the RNA extraction purification column into the tube for the next step.

8. 添加 700µl 洗滌緩衝液 1 to the RNA extraction purification column, 離心機在 14,000 轉速 (20,000×g) 為了 2 分分鐘, extend the 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. 乙醇的存在對隨後的實驗有嚴重影響, 因此膜乾燥至關重要. 離心後, ensure there is no ethanol present before elution. Discard the waste liquid and the collection tube.

After washing with Wash Buffer 1, the membrane on the RNA extraction purification column should only have a slight color. 離心後, carefully remove the RNA extraction purification column without touching the collection tube to ensure no ethanol interference.)

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

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

10. 重複上一步.

(筆記: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.)