細菌ゲノムDNA抽出キット

• 試薬キットのコンポーネント

• ストレージ

この試薬キットは、室温で保管する必要があります (15-25℃) and in dry conditions, 貯蔵寿命があります 12 月. The DNA extraction purification columns can be stored for 1 year in a cool and dry environment. Lysozyme, Proteinase K and RNase A contain preservatives, allowing transportation at room temperature, しかし、長期保管の場合, they should be kept at -20℃.

• 試薬キットを使用するための指示

3.1 This reagent kit is intended for molecular biology research and should not be used for disease diagnosis or treatment.

3.2 Some components in the reagent kit contain irritants. Protective measures such as wearing protective clothing and goggles are recommended.

3.3 During the usage of this reagent kit, a high-speed centrifuge, 水浴 (メタルバス), 渦ミキサー, 無水エタノール, 滅菌脱イオン水, and EP tubes need to be prepared by the user.

• 試薬キットの紹介

This kit provides a rapid and effective purification method for isolating DNA from various bodily fluids and bacterial culture fluids. It utilizes a silicon-based purification column that selectively adsorbs nucleic acids. With specific buffer solutions, bacterial DNA samples can be extracted within 30 分. 精製プロセス全体では、フェノールクロロホルムなどの有毒試薬は必要ありません. The extracted DNA can be directly used for downstream experiments like PCR, サザンブロット, その他.

• 実験原則と手順

• 抽出プロセス

Before Starting the Experiment:

1. Reagent Buffer B and C 低温条件下で沈殿する可能性があります. We recommend heating at 65℃for 5 分. After the precipitate dissolves, it can be used normally.
2. ご使用の前に, 指定された量の無水エタノールを追加します 洗浄バッファー 1as indicated on the bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.
3. 溶出バッファーはaです0.1X TEソリューションcontaining minimal amounts of EDTA. If EDTA might affect subsequent experiments, it is advisable to substitute Elution Buffer with sterile deionized water.
4. サンプル処理:
5. Take around 1 ml of bacterial culture, で遠心分離する 12,000 の回転数 1 分, aspirate the supernatant as much as possible, 追加 200μl of Reagent Buffer Bto the bacterial cell solution. 一般的に, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, 追加 10μl of RNaseA (10mg/ml) 混合物に, incubate at 37°C for 2 minutes with vortexing during the period.
6. If the sample being processed contains Gram-positive bacteria, 追加 10μl of Lysozyme (10 mg/ml)そして 200μl of Reagent Buffer B. 一般的に, residual RNA has minimal impact on downstream experiments. If RNA interference needs to be eliminated, 追加 10μl of RNaseA (10mg/ml) 混合物に, incubate at 37°C for 15-30 minutes with vortexing during the period.
7. 追加 10μl of Proteinase K (10 mg/ml), thoroughly invert and mix, digest at 65°C for 2 分. During this period, invert and mix the sample solution 6-7 times until the sample solution becomes clear after digestion.
8. 追加 200μl of Reagent Buffer Cto the lysate and mix. If a white precipitate appears, it can be left to settle; it won’t affect subsequent experiments once the precipitate disappears.
9. 追加 200μl of ethanol, 十分に混ぜる. Some precipitation might occur but won’t affect subsequent experiments.
10. Transfer the obtained liquid into a DNA extraction purification column, leave at room temperature for 2 分, で遠心分離する 12,000 の回転数 30 秒. Discard the collected waste and reinsert the collection tube into the purification column for the next step.
11. 追加 600洗浄バッファーμl 1, で遠心分離する 12,000 の回転数 30 秒, 廃棄物を捨てます, and reinsert the DNA extraction purification column into the holder.

(注記: Ensure ethanol has been added to Wash Buffer 1.)

7. 追加 500洗浄バッファーμl 1 to the DNA extraction purification column, で遠心分離する 12,000 の回転数 2 分, extending the centrifugation time as needed for a drier membrane.
8. Place the DNA extraction purification column (holder) 新しい遠心チューブに, 開ける, 65°Cで加熱します 2 分. Extend this step as necessary to evaporate ethanol, preventing ethanol residues from affecting downstream experiments.
9. 追加 50-100μLの溶出緩衝液onto the column membrane, で遠心分離する 12,000 の回転数 2 分.

(注記: 1. DNAを溶出します 50 μl of Elution Buffer can increase DNA concentration but reduce the total DNA yield. 2. The eluted DNA from the eluate can be reapplied to the DNA extraction purification column, 再び遠心分離機 12,000 の回転数 2 minutes to enhance DNA yield.)