便または土壌ゲノム DNA 抽出キット

1. 試薬キットのコンポーネント

仕様	50T	100T
猫. いいえ.	SN0241	SN0242
DNA抽出カラム (セット)	50 (セット)	100 (セット)
Humic acid removal reagent I	50ミリリットル	2x50ml
Humic acid removal reagent II	50ミリリットル	2x50ml
Reagent Buffer Ⅳ	30 ミリリットル	2×30 ミリリットル
Reagent Buffer C plus	30 ミリリットル	2×30 ミリリットル
Inhibitor removal buffer	30ミリリットル	2x30ml
Lysozyme	1ミリリットル	2x1ml
プロテイナーゼK	1ミリリットル	2x1ml
RNase A	1ミリリットル	2x1ml
洗浄バッファー 1	15 ミリリットル	2 × 15 ミリリットル
溶出バッファー	20 ミリリットル	2 ×20 ml
取扱説明書	1	1

2. 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, プロテイナーゼK, そして 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℃.

3. 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.

4. 試薬キットの紹介

This kit provides a fast and efficient method for purifying microbial genomic DNA from soil, 糞便, and vomit samples. It is widely used for microbial DNA extraction from soil, 糞便, and vomit.

 

This kit effectively removes sample impurities and inhibitors, reducing inhibitory reactions in downstream experiments such as PCR. 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 PCR, サザンブロット, その他のアプリケーション.

5. 実験原則と手順

6. 抽出プロセス

Before Starting the Experiment:

あ. Reagent Buffer IV 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.

B. 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 1 as indicated on the bottle label. Mark a check on the label to indicate the addition of anhydrous ethanol.

C. 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.

1. サンプル処理 (Inhibitor Removal):

あ. Fecal and Vomit Samples: Add approximately 200mg of the sample to be extracted, 追加 1ml of Humic Acid Removal Reagent I, vortex thoroughly for 1-2 分, で遠心分離する 12,000 の回転数 2 分, 上清を捨てる, 追加 560μl of Buffer IV to the pellet, invert and mix thoroughly, digest at 85℃ for 5 分, invert and mix 6-7 times during digestion, then proceed to step 2.

B. Soil Samples: Weigh approximately 0.1g-0.5g of sieved soil, 追加 500μl of Buffer IV and 100μl of Humic Acid Removal Reagent I, invert and mix thoroughly, digest at 85℃ for 5 分, invert and mix 6-7 times during digestion, then proceed to step 2.

(注記: Humic Acid Removal Reagent I/Humic Acid Removal Reagent II is mainly used for soils with high humic acid content, such as forest soils and sedimentary soils. 追加 1ml of Humic Acid Removal Reagent I to the sample, vortex, shake for 1-2 分, で遠心分離する 8,000 の回転数 2 分, 上清を捨てる, それから追加します 1ml of Humic Acid Removal Reagent II, vortex, で遠心分離する 8,000 の回転数 2 分, 上清を捨てる. This step can further reduce soil humic acid inhibitors but significantly reduce DNA yield.)

2. で遠心分離します。 12,000 の回転数 2 分, transfer the supernatant to a new centrifuge tube (approximately 500μl liquid transferred), add 20μl Proteinase K (10 mg/ml), 20μl Lysozyme, and 20μl RNase A, and incubate at 65℃ for 2 分.
3. 等量を追加します 試薬 Buffer C plus (approximately 560μl liquid transferred) to the lysate, よく混ぜます. If a white precipitate appears, let it settle; it won’t affect subsequent experiments after the precipitate disappears.
4. Add an equal volume of ethanol to 試薬 Buffer C plus (例えば, 追加 560μlの 試薬 Buffer C plus, then add 560μl of ethanol), よく混ぜます. There may be precipitation, but it won’t affect subsequent experiments.
5. Add the obtained liquid to the DNA extraction purification column (sleeve) (毎回約650〜700μl), オーバーでの遠心 8,000 の回転数 1 分, discard the collected waste liquid, and reinsert the collection tube into the DNA extraction purification column (sleeve) for the next step.
6. 追加 400μl Inhibitor Removal Buffer, オーバーでの遠心 8,000 の回転数 1 分, discard the waste liquid, and reinsert the DNA精製 column into the sleeve for the next step.
7. 追加 500μl Wash Buffer 1 to the DNA extraction purification column (sleeve), で遠心分離する 14,000 RPM (20,000×g) のために 1 分.
8. 追加 300μl Wash Buffer 1 again to the DNA extraction purification column (sleeve), で遠心分離する 14,000 RPM (20,000×g) のために 2 分.
9. Place the DNA extraction purification column (sleeve) 新しい遠心チューブに, open the lid, and incubate at 65℃ for 2 分. This step can be extended if necessary to evaporate ethanol as much as possible to prevent ethanol residue from affecting downstream experiments.
10. 滴下 100μl Elution Buffer 膜に, で遠心分離する 12,000 の回転数 2 分.

(注記: 1. Eluting DNA with 50μl Elution Buffer can increase DNA concentration but reduce total DNA yield; 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged again at 12,000 の回転数 2 分, and collected to increase DNA yield.)