基本描述

英文名称

DNA Methylation Kit

储存温度

2-8°C储存,室温

运输条件

冰袋运输

产品介绍

D665729	Component	50 T	Storage
D665729A	Conversion Buffer CR	5×1 mL	RT
D665729B	Buffer CL	30 mL	RT
D665729C	Buffer MD	0.4 mL	RT
D665729D	Buffer DB	10 mL	RT
D665729E	Buffer WB (concentrate)	10 mL	RT
D665729F	Buffer GW1 (concentrate)	13 mL	RT
D665729G	Buffer GW2 (concentrate)	15 mL	RT
D665729H	Buffer EB	4 mL	RT
D665729I	Buffer PS	10 mL	RT
D665729J	Spin Columns DF	50 Pcs	2-8 ℃
D665729K	Collection Tubes	50 Pcs	RT

产品内容：

本试剂盒基本原理为DNA经亚硫酸氢钠处理后，可使未甲基化的胞嘧啶转变为尿嘧啶，而甲基化的胞嘧啶不变。并采用独创高温处理方法，极大的缩短了转变时间，提高了转变效率，转变效率可达到99%以上。同时采用硅基质膜式纯化柱通过简单的结合-洗涤-洗脱步骤，可从甲基化修饰后的溶液中回收纯化DNA，回收的DNA纯度高，完整性好，可直接用于测序、甲基化PCR检测、芯片分析、连接和转化、酶切、标记、显微注射、PCR和体外转录等各种分子生物学实验。

自备试剂：无水乙醇、75%乙醇。

实验前准备及重要注意事项

1．产品配用方法：

（1）10次包装配制方法：CT Conversion Reagent是固体混合物，一定要在首次使用前制备好。将2 ml无菌水、100 μl M-Dissolving Buffer和300 μl M-Dilution Buffer加入到CT Conversion Reagent管中。在55°C溶解并震荡直至全部溶解。在使用之前将 CT Conversion Reagent 溶液在室温(20°C -30°C)下避光保存。每管的CT Conversion Reagent 是针对10次 DNA 处理设计的，为了得到较好的结果, 配置好的CT Conversion Reagent应当立即使用，如果不立即使用，可将CT Conversion Reagent 溶液在-20°C存储1周，使用前，请务必将存储的CT Conversion Reagent 溶液在室温下解冻，并且通过振动或颠倒2分钟以充分混匀，CT Conversion Reagent对光很敏感，所以要尽量减少在光下的暴露。

（2）50次包装配制方法：CT Conversion Reagent、M-Dissolving Buffer均固体混合物，一定要在首次使用前制备好。将5 ml无菌水加入到M-Dissolving Buffer中震荡溶解，待固体全部溶解后将M-Dissolving Buffer管中溶液全部转移至CT Conversion Reagent管中同时补加5.5 ml无菌水。再将1.5 ml M-Dilution Buffer加入到CT Conversion Reagent管中。在55°C溶解并震荡直至全部溶解。在使用之前将 CT Conversion Reagent 溶液在室温 (20°C -30°C)下避光保存。每管的 CT Conversion Reagent 是针对50次 DNA 处理设计的，为了得到较好的结果, CT Conversion Reagent应当在制备后立即使用，如果不立即使用，可将 CT Conversion Reagent 溶液在-20°C存储1周，使用前，请务必将存储的 CT Conversion Reagent 溶液在室温下解冻，并且通过振动或颠倒2分钟以充分混匀，CT Conversion Reagent对光很敏感，所以要尽量减少在光下的暴露。

2．第一次使用前应按照试剂瓶标签的说明先在M-Wash Buffer中加入无水乙醇。

操作步骤

每次制备的DNA 范围在 1 ng-4 μg之间，最佳量为500 ng-2μg。

1．取20 μl DNA样品，加入到离心管（自备）中，如果样品量不足，用水补至20μl。

2．向DNA样品中加入2.2 μl 的 M-Dilution Buffer，混匀样品。

3．42℃水浴30分钟。

4．向上步得到的样品中，加入220 μl配制好的 CT Conversion Reagent溶液，混匀， 80℃恒温水浴锅中避光孵育60分钟。

5．向上步溶液中加入480 μl M- Buffer PA，温和上下颠倒混匀。

6．柱平衡：向已装入收集管的吸附柱（Spin Columns DS）中加入200 μl Buffer PS， 12,000 rpm（~13,400×g）离心2分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

将步骤5所得溶液全部加入到吸附柱（已装入收集管）中，室温放置2分钟，12,000 rpm 离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

注意：吸附柱最大容量为750 μl，若样品体积大于750 μl可分批加入。

8．向吸附柱中加入500 μl M- Buffer PA，12,000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱放回收集管中。

9．向吸附柱中加入650 μl M-Wash Buffer（使用前请先检查是否已加入无水乙醇）， 12,000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱放回收集管中。

10．12,000 rpm离心2分钟，倒掉废液，将吸附柱置于室温数分钟，以彻底晾干。

注意：这一步的目的是将吸附柱中残余乙醇去除，乙醇残留会影响后续的酶促反应（酶切、PCR等）。

11．将吸附柱放入一个新的离心管（自备）中，向吸附膜中间位置悬空滴加20 μl M-Elution Buffer（pH 8.5），室温放置2分钟。12,000 rpm离心1分钟收集DNA溶液。

12．向收集的20 μl DNA中加入2.2 μl M-Dilution Buffer，室温静止30分钟。

13．向溶液中加入500 μl预冷的无水乙醇后颠倒混匀，并将溶液置于-20℃沉淀30分钟（过夜沉淀效果更好）。

14．12,000 rpm离心15分钟，轻轻倒掉上清。

15．加入75%乙醇，12,000 rpm离心1分钟，倒掉上清，室温下待乙醇挥发后，加入20 μl M-Elution Buffer溶解，DNA置于-20℃保存。此步收集的DNA可以用于后续相关实验。

D665729	Component	50 T	Storage
D665729A	Conversion Buffer CR	5×1 mL	RT
D665729B	Buffer CL	30 mL	RT
D665729C	Buffer MD	0.4 mL	RT
D665729D	Buffer DB	10 mL	RT
D665729E	Buffer WB (concentrate)	10 mL	RT
D665729F	Buffer GW1 (concentrate)	13 mL	RT
D665729G	Buffer GW2 (concentrate)	15 mL	RT
D665729H	Buffer EB	4 mL	RT
D665729I	Buffer PS	10 mL	RT
D665729J	Spin Columns DF	50 Pcs	2-8 ℃
D665729K	Collection Tubes	50 Pcs	RT

Product Introduction:
The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.
Self prepared reagents: anhydrous ethanol, 75% ethanol.
Preparation and important precautions before the experiment
1. Product usage method:
(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 μ M-Dissolving Buffer and 300 μ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.

(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.
2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.
Operation steps
The range of DNA prepared each time is 1 ng-4 μ Between g, the optimal amount is 500 ng-2 μ G.
1. Take 20 μ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 μ L.
2. Add 2.2 to the DNA sample μ Mix the sample well with the M-Dilution Buffer of l.
3.42 ℃ water bath for 30 minutes.
4. Add 220 to the sample obtained from the previous step μ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.
5. Add 480 to the solution in the previous step μ M - Buffer PA, gently mix upside down.
6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube μ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
Attention: The maximum capacity of the adsorption column is 750 μ l. If the sample volume is greater than 750 μ L can be added in batches.
8. Add 500 to the adsorption column μ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.
9. Add 650 to the adsorption column μ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.
10.12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column at room temperature for a few minutes to thoroughly air dry.
Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air μ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.
12. Collect 20 μ Add 2.2 to DNA μ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.
13. Add 500 to the solution μ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).
14.12000 rpm for 15 minutes and gently discard the supernatant.

15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 μ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

d665729_dna methylation kit.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

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DNA甲基化试剂盒

库存信息

基本描述

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

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