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DNA凝胶回收试剂盒

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D598099-10T
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PCR清洁及胶回收 (1) 核酸提取与纯化 (27)
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基本描述

英文名称 DNA gel Recovery Kit
储存温度 室温
运输条件 常规运输
产品介绍

本试剂盒适合从各种琼脂糖凝胶中回收多至8μgDNA(70bp-10Kb)，回收率为60-85%。琼脂糖凝胶在温和的缓冲液(DE-A溶液)中熔化，其中的保护剂能防止线状DNA在高温下降解，然后在DE-B溶液的作用下使DNA选择性结合到膜上。纯化的DNA纯度高，并保持片断完整性和高生物活性，可直接用于连接、体外转录、PCR 扩增、测序、微注射等分子生物学实验

组分：


组分备注：
Buffer DE-A: 凝胶熔化剂，含DNA保护剂，防止DNA在高温下降解，室温密闭贮存；
Buffer DE-B: 结合液（促使大于70bp的DNA片段选择性结合到DNA制备膜上），室温密闭贮存；
Buffer W1: 洗涤液，室温密闭贮存；
Buffer W2 concentrate: 去盐液，使用前，按试剂瓶上指定的体积加入无水乙醇（用100%乙醇或95%乙醇），混合均匀，室温密闭贮存；
Eluent: 洗脱液，室温密闭贮存。

注意事项：

1.Buffer DE-A(含有 -基醇)Buffer DE-B 和 Buffer W1 含刺激性化合物，操作时要戴乳胶手套和眼镜，避免沾染皮肤、眼睛和衣服，谨防吸入口鼻。若沾染皮肤、眼睛时，要立即用大量清水或生理盐水冲洗，必要时寻求医疗咨询；

2.在步骤 1 中，将凝胶切成细小的碎块可大大缩短凝胶熔化时间(线型 DNA 长时间暴露在高温条件2.下易于水解)，从而提高回收率。勿将含 DNA 的凝胶长时间地暴露在紫外灯下，减少紫外线对 DNA造成的损伤。

3.在步骤 2中凝胶必须完全熔化，否则将严重影响 DNA 回收率；

4.将 Eluent 或去离子水加热至65°C，有利于提高洗脱效率；

5.DNA 分子呈酸性，建议在 2.5mM Tris-HCl，pH 7.0-8.5洗脱液中保存。

实验准备：
1.第一次使用前，Buffer W2 concentrate中加入指定体积的无水乙醇。
2.准备无核酸和核酸酸污染的Tip头、离心管
3.准备75°C水浴。
4.使用前，检查Buffer DE-B是否出现沉淀，若出现沉淀，应于70°C温浴加热熔化并冷却至室温后再使用。

操作步骤：
1.在紫外灯下切下含有目的 DNA 的琼脂糖凝胶，用纸巾吸尽凝胶表面液体并切碎。计算凝胶重量(提前记录 1.5 ml 离心管重量)，该重量作为一个凝胶体积(如 100 mg=100 μl 体积)。
2.加入3个凝胶体积的 Buffer DE-A，混合均后于75C 加热(低熔点琼脂糖凝胶于 40 加热),间断混合(每2-3 min)，直至凝胶块完全熔化(约6-8 min)Buffer DE-A 为红色溶液。在熔化凝胶的过程中，可以帮助观察凝胶是否完全熔化。
3.加0.5个 Buffer DE-A 体积的 Buffer DE-B，混合均。当分离的DNA 片段小于 400bp 时，需再加入1个凝胶体积的异丙醇。
*加 Buffer DE-B 后混合物颜色变为黄色，充分混匀以保证形成均一的黄色溶液。
步骤 4-6 可以选择负压法或离心法。
A.负压法
4A.正确连接负压装置，将 DNA 制备管插到负乐装置的插口上。吸取步 3 中的混合液，转移到制备管中，开启并调节负压至-25-30 英寸汞柱，缓慢吸走管中溶液。
5A.加500 l Buffer W1，吸尽管中溶液。
6A.加700 ul Buffer W2，吸尽管中溶液。以同样的方法再用 700 l Buffer W2洗涤一次，确认在 Buffer W2 concentrate 中已按试剂瓶上的指定体积加入无水乙醉。
·两次使用 Buffer W2 冲洗能确保盐份被完全清除，消除对后续实验的影响。
7A.将制备管置于2ml 离心管 (试剂盒提供)中，12,000xg 离心1min。

B，离心法
4B.吸取步骤3 中的混合液，转移到 DNA 制备管(置于 m 离心管(试剂内提供))中，12000Xg 离心1min。弃滤液。
5B.将制备管置回2 ml离心管，加500 l Buffer W1，12,000Xg 离心30 s，弃滤液。
6B.将制备管置回2 ml 离心管，加 700 l Buffer W2，12,000Xg 离心30s，弃滤液。以同样的方法再用 700 ul Buffer W2 洗涤一次 12,000xg 离心1min。，确认在 Buffer W2 concentrate 中已按过剂瓶上的指定体积加入无水乙醉两次使用 Buffer W2 冲洗能确保盐份被完全清除，消除对后续实验的影响。
7B，将制备管置回2ml 离心管中，12,000Xg 离心1min。
8.将制备管置于洁净的 1.5 m 离心管中，在制备膜中央加 25-30 Euent 或去离子水，室温静置1min。12,000xg 离心1min 洗脱DNA。

将 Eluent 或去离子水加热至 65C将提高洗脱效率


应用范围：

核酸提取及纯化

This kit is suitable for recovering up to 8 μ GDNA (70bp-10kb), the recovery was 60-85%. The agarose gel melts in a mild buffer (De-a solution), in which the protective agent can prevent the degradation of linear DNA at high temperature, and then selectively bind DNA to the membrane under the action of de-b solution. The purified DNA has high purity and maintains fragment integrity and high biological activity, and can be directly used in molecular biology experiments such as ligation, in vitro transcription, PCR amplification, sequencing, microinjection, etc

Component：

Component remarks:

Buffer DE-A : gel melting agent, containing DNA protective agent, preventing DNA degradation at high temperature, sealed storage at room temperature ;
Buffer DE-B : binding solution ( to promote the selective binding of DNA fragments greater than 70bp to DNA preparation membrane ), sealed storage at room temperature ;
Buffer W1 : washing liquid, sealed storage at room temperature ;
Buffer W2 concentrate : desalted liquid, before use, add anhydrous ethanol ( 100 % ethanol or 95 % ethanol ) according to the volume specified on the reagent bottle, mix evenly, and store in airtight at room temperature ;
Eluent : eluent, sealed storage at room temperature.

Points for attention：

1.Buffer DE-A ( containing-alkyl alcohol ) Buffer DE-B and Buffer W1 contain irritating compounds. Wear latex gloves and glasses during operation to avoid contamination of skin, eyes and clothes, and beware of inhalation of mouth and nose. If the skin, eyes, to immediately rinse with a lot of water or saline, if necessary, seek medical advice ; 

2.In step 1, cutting the gel into small pieces can greatly shorten the melting time of the gel ( linear DNA is easily hydrolyzed under high temperature conditions for a long time ), thereby increasing the recovery rate. Do not expose the gel containing DNA to ultraviolet light for a long time to reduce the damage caused by ultraviolet light to DNA. 

3.In step 2, the gel must be completely melted, otherwise it will seriously affect the DNA recovery rate ; 

4.Heating Eluent or deionized water to 65 ° C is beneficial to improve the elution efficiency ; 

5.DNA molecules are acidic, and it is recommended to preserve in 2.5mM Tris-HCl, pH 7.0-8.5 eluent.

Experimental preparation:
Before the first use, add a specified volume of anhydrous ethanol to Buffer W2 concentrate.
2. Prepare tip heads and centrifuge tubes free from nucleic acid and nucleic acid contamination
3. Prepare a 75 ° C water bath.
4. Before use, check if there is any precipitation in Buffer DE-B. If precipitation occurs, it should be heated and melted in a 70 ° C temperature bath and cooled to room temperature before use.

Operation steps:
1. Cut the agarose gel containing the target DNA under the UV lamp, suck up the liquid on the surface of the gel with a paper towel and cut it into pieces. Calculate the weight of gel (record the weight of 1.5 ml centrifuge tube in advance), which is taken as a volume of gel (e.g. 100 mg=100 μ L volume).
2. Add three gel volumes of Buffer DE-A, heat them at 75C after mixing (low melting point agarose gel is heated at 40), intermittently mix them (every 2-3 min) until the gel block is completely melted (about 6-8 min), and Buffer DE-A is a red solution. In the process of melting gel, it can help to observe whether the gel is completely melted.
3. Add 0.5 buffer DE-A volumes of buffer DE-B and mix evenly. When the isolated DNA fragment is less than 400 bp, one more gel volume of isopropanol shall be added.
*After adding Buffer DE-B, the color of the mixture turns yellow. Mix thoroughly to ensure the formation of a uniform yellow solution.
Steps 4-6 can choose between negative pressure method or centrifugal method.
A. Negative pressure method
4A. Connect the negative pressure device correctly and insert the DNA preparation tube into the socket of the negative pressure device. Suck the mixture from step 3, transfer it to the preparation tube, open and adjust the negative pressure to -25-30 inches of mercury, and slowly remove the solution from the tube.
5A. Add 500 l of Buffer W1 and absorb the intermediate solution.
6A. Add 700 ul Buffer W2 and absorb the intermediate solution. Wash again with 700 l of Buffer W2 using the same method, and confirm that anhydrous ethanol has been added to the buffer W2 concentrate according to the specified volume on the reagent bottle.
·Two washes with Buffer W2 can ensure complete removal of salt and eliminate any impact on subsequent experiments.
7A. Place the preparation tube in a 2ml centrifuge tube (provided by the reagent kit) and centrifuge 12000xg for 1 minute.

B. Centrifuge method
4B. Take the mixture from step 3 and transfer it to a DNA preparation tube (placed in an m centrifuge tube (provided in the reagent). Centrifuge at 12000Xg for 1 minute. Discard the filtrate.
5B. Place the preparation tube back into a 2 ml centrifuge tube, add 500 l Buffer W1, centrifuge 12000 Xg for 30 seconds, and discard the filtrate.
6B. Place the preparation tube back into a 2 ml centrifuge tube, add 700 l Buffer W2, centrifuge 12000 Xg for 30 seconds, and discard the filtrate. Using the same method, wash once with 700 ul Buffer W2 and centrifuge 12000xg for 1 minute., Confirm that anhydrous ethyl alcohol has been added to the designated volume on the vials in Buffer W2 concentrate twice. Rinsing with Buffer W2 can ensure complete removal of salt and eliminate any impact on subsequent experiments.
7B, place the preparation tube back into a 2ml centrifuge tube and centrifuge 12000 Xg for 1 minute.
8. Place the preparation tube in a clean 1.5m centrifuge tube, add 25-30 Euent or deionized water in the center of the preparation membrane, and let it stand at room temperature for 1 minute. Wash DNA by centrifugation at 12000xg for 1 minute.
Heating Elent or deionized water to 65C will improve the elution efficiency.


Scope of application：

Nucleic acid extraction and purification

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