Paraffin-embedded Tissue DNA Extraction Kit

1. 試薬キットのコンポーネント

仕様	50T	100T
猫. いいえ.	SN0251	SN0252
DNA抽出カラム (セット)	50 (セット)	100 (セット)
Reagent Buffer IV	20 ミリリットル	2 × 20 ミリリットル
Reagent Buffer C	30 ミリリットル	2 × 30ml
Reagent Buffer FF	60 ミリリットル	2 × 60ml
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	20 ミリリットル
プロテイナーゼK	1ミリリットル	2x1ml
RNaseA	1ミリリットル	2x1ml
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. 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℃.

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

 

Paraffin-embedded tissue DNA精製 kit provides a rapid and effective DNA purification solution for samples in paraffin sections. Tissues are collected after paraffin dissolution using a dedicated dewaxing solution, and sample DNA is obtained through subsequent extraction processes.

 

This kit can extract sample DNA from paraffin sections within 30 分, and the entire purification process does not require toxic reagents such as phenol-chloroform. 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 FF:This buffer should be stored long-term in an environment between 2°C and 8°C.

B. Reagent Buffers IV and C 低温条件下で沈殿する可能性があります. 65°Cで加熱することをお勧めします 5 分. After the precipitate dissolves, the solution can be used normally.

C. ウォッシュバッファ 1 should have a specified amount of anhydrous ethanol added before use, as indicated on the reagent bottle label. Check the label with a tick mark to indicate the addition of anhydrous ethanol.

D. 溶出バッファーはaです 0.1X TEソリューション 最小限のEDTAで. EDTAがその後の実験に影響を与える場合, it is advisable to replace the elution buffer with sterile deionized water.

 

1. サンプル処理:

Select 5-10 paraffin sections (1mm-3mm thickness), use scissors to remove excess wax, and cut the embedded tissue sample into small pieces. Place them in a 1.5ml centrifuge tube, 追加 800μl Reagent Buffer FF, incubate at 75°C for 5 minutes until all paraffin is dissolved. Centrifuge at 12000rpm for 5 分, 上清を捨てる.

2. 追加 400μl Reagent Buffer IV, 20μl Proteinase K (10 mg/ml), and 10μl RNaseA (10 mg/ml) to the precipitate from the previous step. Digest at 65°C, stirring every 6-7 times until digestion is complete.
3. 等量を追加します Reagent Buffer Cそして an equal volume of anhydrous ethanol, mix thoroughly by pipetting.

(例えば: If you add 400μl Reagent Buffer IV, approximately add 430μl Reagent Buffer C and 430μl anhydrous ethanol. Some precipitation may occur after adding Reagent Buffer C, but it does not affect subsequent experiments.)

4. Add the obtained liquid to the DNA extraction purification column (or cartridge) (毎回約650〜700μl), let it stand at room temperature for 2 分, centrifuge at over 8,000rpm for 1 分. Discard the collected waste and reinsert the collection tube into the purification column for the next step.
5. ステップを繰り返します 4, adding the remaining liquid to the DNA extraction purification column (or cartridge), centrifuge at over 8,000rpm for 1 分, discard the waste and the collection tube.
6. Place the DNA extraction purification column (or cartridge) into the collection tube, 追加 300μl ウォッシュバッファ 1, centrifuge at over 8,000rpm for 1 分. Discard the waste and reinsert the purification column into the tube for the next step.

(注記: Confirm that anhydrous ethanol has been added to ウォッシュ バッファ 1.)

7. 追加 500μl ウォッシュバッファ 1 to the DNA extraction purification column (or cartridge), centrifuge at 14,000rpm (20000×g) のために 2 分. Extend centrifugation time if needed for a dryer membrane.
8. Place the DNA extraction purification column (or cartridge) in a new centrifuge tube, open the lid, incubate at 65°C for 2 分. Extend this step if necessary to evaporate ethanol to prevent residual ethanol from affecting downstream experiments.
9. Suspend the addition of 50-100μl Elution Buffer 膜に, centrifuge at 12,000rpm for 2 分.

(注記: 1. Using 50μl Elution Buffer for DNA elution can increase DNA concentration but decreases total DNA yield. 2. The eluted DNA can be reapplied to the DNA extraction purification column, centrifuged at 12000rpm for 2 minutes again to increase DNA yield.)