1. 試劑盒的組成
| 規格 | 50時間 | 100時間 |
| 貓. 不. | SN0253 | SN0254 |
| DNA萃取柱 (放) | 50 (放) | 100 (放) |
| Reagent Buffer SP | 20 毫升 | 2 × 20 毫升 |
| 試劑緩衝液C | 30 毫升 | 2 × 30ml |
| 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
The sperm sample genomic DNA extraction kit provides a rapid and efficient purification solution for sperm sample DNA. It achieves sample DNA純化 effectively through a dedicated extraction buffer system combined with specific nucleic acid purification columns.
This kit can extract sperm sample DNA 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 SP:This buffer should be stored long-term in an environment between 2℃ and 8℃
乙.試劑緩衝液C 低溫條件下可能會沉澱. It is recommended to heat at 65℃ for 5 分分鐘. 沉澱溶解後, 可以正常使用.
C.洗滌緩衝液 1: 使用前, add the specified amount of anhydrous ethanol as indicated on the reagent bottle label. 在標籤上打勾，表示添加了無水乙醇.
D. 洗脫緩衝液是 0.1x TE 解決方案 含有極少量的 EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water as a substitute for the elution buffer.
- 樣品加工:
吸管 200微升 of frozen sperm, incubate at 75℃ for 10 minutes until the sperm sample is not viscous. 離心機在 12000 轉速為 5 分分鐘, aspirate the supernatant as much as possible, and the sperm cells will sediment at the bottom of the tube.
- Add to the sediment from the previous step: 400μl Reagent Buffer SP, 20μl Proteinase K (10 毫克/毫升), 20µl RNaseA (10 毫克/毫升).Digest at 65℃ for 10 分分鐘, 反轉並混合 6-7 times during digestion until the sample is fully digested.
- 加入等體積的 試劑緩衝液C and an equal volume of anhydrous ethanol, and mix well by pipetting.
(例如: If you add 400μl of Reagent Buffer IV, you will need to add approximately 460μl of Reagent Buffer C and 460μl of anhydrous ethanol. A small amount of precipitation may form after adding Reagent Buffer C, but it does not affect subsequent experiments.)
- Transfer the obtained liquid to a DNA extraction purification column (cassette) (每次約650-700μl). Let it stand at room temperature for 2 分分鐘, 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.
- 重複步驟 4, adding the remaining liquid to the DNA extraction purification column (cassette), 離心機超過 8,000 轉速為 1 分分鐘, discard the waste liquid and the collection tube.
- 放置DNA萃取純化管柱 (cassette) 進入收集管, 添加 300 µl 洗滌緩衝液 1, 離心機超過 8,000 轉速為 1 分分鐘, 丟棄廢液, 並放置DNA萃取純化管柱 (cassette) 放回管中進行下一步.
(筆記: Confirm that anhydrous ethanol has been added to Wash Buffer 1.)
- 添加 500µl 洗滌緩衝液 1 至DNA萃取純化管柱 (cassette), 離心機在 14,000 轉速 (20,000×g) 為了 2 分分鐘, extend the centrifugation time appropriately to dry the membrane more thoroughly.
- 放置DNA萃取純化管柱 (cassette) 放入新的離心管中, 打開蓋子, incubate at 65℃for 2 分分鐘. Extend this step if necessary to evaporate ethanol as much as possible, preventing ethanol residue from affecting downstream experiments.
- Drop-suspend 50-100µl 洗脫緩衝液 到膜上, 離心機在 12,000 轉速為 2 分分鐘.
(筆記: 1. Using 50μl of Elution Buffer to elute DNA can increase DNA concentration but decreases the overall DNA yield. 2. 洗脫的 DNA 可重新上樣至 DNA 萃取純化柱, centrifuged at 12,000 轉速為 2 minutes again, to increase DNA yield.)
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