基本描述

英文名称

AllPure DNA/RNA/Protein Kit

储存温度

室温

运输条件

常规运输

产品介绍

本试剂盒适用于从同一细胞或组织样本中同时分离纯化基因组DNA、总RNA和总蛋白。本产品无需将样品分为3份来分别提取DNA，RNA和蛋白质，也无需先将纯化后的总核酸分为两份后再分别纯化DNA和RNA，因此可最大限度的回收DNA，RNA和蛋白质，可以用于小量样品、珍稀样品的核酸和蛋白纯化。纯化后的DNA、RNA和蛋白质可被单独洗脱，可直接应用于下游多种分子生物学操作。本试剂盒中不包含苯酚和氯仿等有毒物质，无需乙醇沉淀，操作简单、快捷。提取的基因组DNA可用于PCR、Real-time PCR、SouthernBlot、Dot Blot、比较基因组杂交（CGH）、基因分析和SNP分析；总RNA可应用于RT-PCR、cDNA的合成、Nothern Blot、Dot Blot和基因芯片等实验；总蛋白可应用于电泳和WesternBlot等。

A665492	Component	50 T	Storage
A665492A	Buffer RL	35 mL	RT
A665492B	Buffer RW1	40 mL	RT
A665492C	Buffer RW2 (concentrate)	11 mL	RT
A665492D	RNase-Free Water	10 mL	RT
A665492E	Buffer GW1 (concentrate)	13 mL	RT
A665492F	Buffer GW2 (concentrate)	15 mL	RT
A665492G	Buffer GE	15 mL	RT
A665492H	Buffer PZ	60 mL	RT
A665492I	Buffer PLS	15 mL	RT
A665492J	Spin Columns DM with Collection Tubes	50 sets	RT
A665492K	Spin Columns RM with Collection Tubes	50 sets	RT
A665492L	Collection Tubes	100 EA	RT
A665492M	RNase-Free Centrifuge Tubes (1.5 mL)	100 EA	RT

自备试剂：

β-巯基乙醇（新开封或提取RNA专用）、70%乙醇（用无RNase的水配制）、无水乙醇。

实验前准备及重要注意事项：

1．预防RNase污染，应注意以下几方面：

1 ) 使用无RNase的塑料制品和枪头，避免交叉污染。

2 ) 玻璃器皿应在使用前于180℃高温下干烤4小时，塑料器皿可在0.5 M NaOH中浸泡10分钟，用水彻底冲洗后高压灭菌。

3 ) 配制溶液应使用无RNase的水。

4 ) 操作人员戴一次性口罩和手套，实验过程中要勤换手套。

2．样品应避免反复冻融，否则影响提取DNA、RNA和蛋白质的质量。样品在Buffer RL 中，可于-70℃保存一个月。

3．Buffer RL在使用前请加入β-巯基乙醇，1 ml Buffer RL加10 μl β-巯基乙醇。加入β-巯基乙醇的Buffer RL室温可保存1个月。

4．第一次使用前应按照试剂瓶标签的说明在Buffer RW2、Buffer GW1和Buffer GW2中加入无水乙醇。

5．使用前请检查Buffer RL是否出现结晶或者沉淀，如有结晶或者沉淀，请置于56℃水浴重新溶解。

6．所有离心步骤均使用台式离心机，室温下进行。

操作步骤：

1．材料处理

1a. 贴壁培养的细胞应先处理为细胞悬液（最大提取量为107 个细胞）, 收集细胞，弃尽培养液，加入600 μl Buffer RL（用前检查是否已加入β-巯基乙醇），反复吹打使其充分裂解。

注意：一定要弃尽培养液，否则影响裂解和后续的核酸纯化步骤。

1b. 取不大于30 mg的动物组织，液氮研磨成细粉末，加入600 μl Buffer RL（用前检查是否加入β-巯基乙醇），或直接加入600 μl Buffer RL（使用前检查是否已加入β-巯基乙醇），匀浆处理。

注意：匀浆要充分，否则影响RNA产量。

2．将上步所得溶液12,000 rpm（~13,400×g）离心3-5分钟，小心的将上清加入到已装入收集管吸附柱DM（Spin Columns DM）中，12,000 rpm离心30-60秒，收集滤液。将吸附柱DM放在一个新的2 ml收集管中，室温或4℃放置，待提DNA。注意：确保吸附柱上没有液体残留，如果有必要可以重复离心，直至所有的液体通过吸附柱的膜。总RNA提取

3．向步骤2所得滤液中加入1倍体积的70%乙醇（无RNase水配制），混匀。

4．将上步所得溶液全部加入到已装入收集管的吸附柱（Spin Columns RM）中，若一次不能全部加完溶液，可分次转入。12,000 rpm离心20秒，保留收集管中的液体，用于蛋白提取。

5．将吸附柱RM放入一个新的2ml收集管中，向吸附柱RM中加入700 μl Buffer RW1, 12,000 rpm离心20秒，倒掉收集管中的废液，将吸附柱RM放回收集管中。

6．向吸附柱RM中加入500 μl Buffer RW2（使用前检查是否已加入无水乙醇）,12,000 rpm 离心20秒, 倒掉收集管中的废液，将吸附柱RM放回2 ml收集管中。

7．重复步骤6。

8．12,000 rpm离心2分钟，倒掉收集管中的废液。将吸附柱置于室温数分钟，以彻底晾干。

注意：此步骤目的是去除吸附柱中残余的乙醇，乙醇的残留会影响后续的酶促反应（酶切、PCR等）。

9．将吸附柱RM置于一个新的无RNase的1.5 ml离心管中，向吸附柱RM的中间部位加入 30-50 μl RNase-Free Water，室温放置2-5分钟，12,000 rpm离心1分钟，收集RNA溶液，-70℃保存RNA，防止降解。

注意：

1）RNase-Free Wate体积不应小于30 μl，体积过小影响回收率。

2）如果要提高RNA的产量，可用30-50 μl新的RNase-Free Water重复步骤9。

3）如果要提高RNA浓度，可将得到的溶液重新加入到吸附柱中，重复步骤9。

基因组DNA提取

10.向吸附柱DM中加入500 μl Buffer GW1（使用前检查是否已加入无水乙醇）, 12,000 rpm 离心20秒，倒掉收集管中的废液，将吸附柱DM放回收集管中。

11.向吸附柱 DM中加入500 μl Buffer GW2（使用前检查是否已加入无水乙醇）, 12,000 rpm 离心2分钟，倒掉收集管中的废液，将吸附柱DM放回收集管中。

注意：如需进一步提高DNA纯度，可重复步骤11。

12.12,000 rpm离心2分钟，倒掉收集管中的废液。将吸附柱DM置于室温数分钟，以彻底晾干吸附柱中的乙醇。

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

13.将吸附柱DM置于一个新的离心管中，向吸附柱DM的中间部位悬空加入100 μl Buffer GE ,室温放置2-5分钟，12,000 rpm离心2分钟，收集DNA溶液，-20℃保存DNA。

注意：

1）Buffer GE的体积不应小于100 μl，体积过小影响回收率。

2）如果要提高DNA的产量，将100 μl新的Buffer GE加至吸附柱上，重复步骤13；如果要提高DNA 浓度，可以将步骤13所得的DNA洗脱液重新加至吸附柱上，重复步骤13。

蛋白质提取

14.在提取RNA流出液（即步骤4所得溶液）中加入1倍体积的Buffer PZ，充分混匀，室温放置10-30分钟。

15.12,000 rpm离心10分钟，弃上清。

16.加入500 μl 70%乙醇，12,000 rpm离心1分钟，尽可能吸尽上清。

17.将离心管置于室温数分钟，以晾干沉淀。

注意：这一步的目的是将残余的乙醇去除，过分干燥会使蛋白沉淀难于溶解，干燥不彻底残留的乙醇会影响蛋白上样。

18.加入100 μl Buffer PLS，得到蛋白溶液。

注意：

1）采用Buffer PLS溶解得到的蛋白样品适用于SDS-PAGE和Western Blot检测，但不适用于Bradford方法进行蛋白定量，如需采用Bradford方法进行蛋白定量可采用5%SDS溶解蛋白，或根据下游试验选择合适的蛋白溶解缓冲液。

2）加入溶解蛋白缓冲液的量根据初始样本量及下游试验具体要求来确定。

3）溶解的蛋白可置于-20℃保存数月，置于2-8℃保存数天。

蛋白样品如需进行SDS-PAGE电泳可进行如下操作：

19.蛋白样品中加入蛋白Loading Buffer，95℃变性5-10分钟，将样品冷却至室温。 20.12,000 rpm离心1分钟，吸取上清进行下游的SDS-PAGE或Western blot等试验。

This reagent kit is suitable for simultaneously isolating and purifying genomic DNA, total RNA, and total protein from the same cell or tissue sample. This product does not require dividing the sample into three parts to extract DNA, RNA, and protein separately, nor does it require dividing the purified total nucleic acid into two parts before purifying DNA and RNA separately. Therefore, it can maximize the recovery of DNA, RNA, and protein, and can be used for the purification of nucleic acid and protein in small and rare samples. The purified DNA, RNA, and protein can be eluted separately and directly applied to various downstream molecular biology operations. This reagent kit does not contain toxic substances such as phenol and chloroform, and does not require ethanol precipitation. The operation is simple and fast. The extracted genomic DNA can be used for PCR, Real time PCR, SouthBlot, Dot Blot, comparative genomic hybridization (CGH), gene analysis, and SNP analysis; Total RNA can be applied in experiments such as RT-PCR, cDNA synthesis, Northern Blot, Dot Blot, and gene chips; Total protein can be applied in electrophoresis and Western Blot, among others.

A665492	Component	50 T	Storage
A665492A	Buffer RL	35 mL	RT
A665492B	Buffer RW1	40 mL	RT
A665492C	Buffer RW2 (concentrate)	11 mL	RT
A665492D	RNase-Free Water	10 mL	RT
A665492E	Buffer GW1 (concentrate)	13 mL	RT
A665492F	Buffer GW2 (concentrate)	15 mL	RT
A665492G	Buffer GE	15 mL	RT
A665492H	Buffer PZ	60 mL	RT
A665492I	Buffer PLS	15 mL	RT
A665492J	Spin Columns DM with Collection Tubes	50 sets	RT
A665492K	Spin Columns RM with Collection Tubes	50 sets	RT
A665492L	Collection Tubes	100 EA	RT
A665492M	RNase-Free Centrifuge Tubes (1.5 mL)	100 EA	RT

Self prepared reagents:
β- Mercaptoethanol (for newly opened or RNA extraction), 70% ethanol (prepared with water without RNase), and anhydrous ethanol.

Preparation and important precautions before the experiment:
To prevent RNase pollution, attention should be paid to the following aspects:
1) Use plastic products and gun heads without RNase to avoid cross contamination.
2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.
3) The solution should be prepared using water without RNase.
4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
2. The sample should avoid repeated freeze-thaw cycles, otherwise it will affect the quality of DNA, RNA, and protein extraction. The sample can be stored in Buffer RL at -70 ℃ for one month.
3. Please add Buffer RL before use β- Mercaptoethanol, 1 ml Buffer RL with 10 μ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.
Before the first use, anhydrous ethanol should be added to Buffer RW2, Buffer GW1, and Buffer GW2 according to the instructions on the reagent bottle label.
5. Before use, please check if there is any crystallization or precipitation in the Buffer RL. If there is any crystallization or precipitation, please dissolve it again in a 56 ℃ water bath.
6. All centrifugation steps are performed using a desktop centrifuge at room temperature.

Operation steps:
1. Material processing
1a The cells cultured on the wall should be first processed into cell suspension (maximum extraction amount of 107 cells), collected cells, discarded the culture medium, and added 600 cells μ L Buffer RL (check if it has been added before use) β- Mercaptoethanol), repeatedly blow and beat to fully decompose.
Attention: It is necessary to discard the culture medium completely, otherwise it will affect the lysis and subsequent nucleic acid purification steps.
1b Take no more than 30 mg of animal tissue, grind it into fine powder with liquid nitrogen, and add 600 μ Buffer RL (check if it has been added before use) β- Mercaptoethanol, or directly add 600 μ L Buffer RL (check if it has been added before use) β- Mercaptoethanol, homogenization treatment.
Attention: The homogenate should be sufficient, otherwise it will affect RNA production.
2. Centrifuge the solution obtained in the previous step at 12000 rpm (~13400 × g) for 3-5 minutes. Carefully add the supernatant to the spin columns DM that have been loaded into the collection tube. Centrifuge at 12000 rpm for 30-60 seconds and collect the filtrate. Place the adsorption column DM in a new 2 ml collection tube at room temperature or 4 ℃ for DNA extraction. Attention: Ensure that there is no liquid residue on the adsorption column, and if necessary, repeat centrifugation until all liquids pass through the membrane of the adsorption column. Total RNA extraction
3. Add 1 volume of 70% ethanol (prepared without RNase water) to the filtrate obtained in step 2, and mix well.
4. Add all the solution obtained in the previous step to the spin columns RM that have been loaded into the collection tube. If the solution cannot be added completely at once, it can be transferred in stages. Centrifuge at 12000 rpm for 20 seconds and retain the liquid in the collection tube for protein extraction.
5. Place the adsorption column RM into a new 2ml collection tube and add 700 to the adsorption column RM μ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM into the recovery manifold.
6. Add 500 to the adsorption column RM μ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the 2 ml collection tube.
7. Repeat step 6.
Centrifuge at 8.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.
Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
9. Place the adsorption column RM in a new 1.5 ml centrifuge tube without RNase, and add 30-50 to the middle of the adsorption column RM μ Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.
Attention:
1) The volume of RNase Free Water should not be less than 30 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 30-50 μ Repeat step 9 for the new RNase Free Water.
3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 9.

Genomic DNA extraction
10. Add 500 to the adsorption column DM μ Buffer GW1 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube.
11. Add 500 to the adsorption column DM μ Buffer GW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column DM into the recovery tube.
Attention: To further improve DNA purity, repeat step 11.
Centrifuge at 12.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column DM at room temperature for a few minutes to thoroughly dry the ethanol in the column.
Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
13. Place the adsorption column DM in a new centrifuge tube and add 100 to the middle of the adsorption column DM by suspending it in the air μ L Buffer GE, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, collect DNA solution, and store DNA at -20 ℃.
Attention:
1) The volume of Buffer GE should not be less than 100 μ l. Small volume affects the recovery rate.
2) If we want to increase DNA production, we will μ Add a new Buffer GE to the adsorption column and repeat step 13; If you want to increase the DNA concentration, you can add the DNA eluent obtained in step 13 back onto the adsorption column and repeat step 13.

Protein extraction
14. Add 1 volume of Buffer PZ to the RNA extraction effluent (i.e. the solution obtained in step 4), mix well, and let it stand at room temperature for 10-30 minutes.
Centrifuge at 15.12000 rpm for 10 minutes and discard the supernatant.
16. Add 500 μ Centrifuge at 12000 rpm for 1 minute with 70% ethanol, and try to absorb the supernatant as much as possible.
17. Place the centrifuge tube at room temperature for a few minutes to dry the precipitate.
Attention: The purpose of this step is to remove residual ethanol. Excessive drying can make protein precipitation difficult to dissolve, and incomplete drying of residual ethanol can affect protein loading.
18. Add 100 μ L Buffer PLS to obtain protein solution.
Attention:
1) The protein samples obtained by dissolving with Buffer PLS are suitable for SDS-PAGE and Western Blot detection, but not for Bradford method for protein quantification. If Bradford method is needed for protein quantification, 5% SDS can be used to dissolve the protein, or suitable protein dissolution buffer can be selected based on downstream experiments.
2) The amount of dissolved protein buffer added is determined based on the initial sample size and specific downstream test requirements.
3) The dissolved protein can be stored at -20 ℃ for several months and at 2-8 ℃ for several days.
If protein samples require SDS-PAGE electrophoresis, the following operations can be performed:
19. Add protein loading buffer to the protein sample, denature at 95 ℃ for 5-10 minutes, and cool the sample to room temperature. Centrifuge at 20.12000 rpm for 1 minute, extract the supernatant for downstream SDS-PAGE or Western blot tests.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

a665492_allpure dnarnaprotein kit.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

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

输入批号以搜索分析图谱:

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

=

浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

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浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

÷

所需的复溶浓度

DNA/RNA/Protein 提取试剂盒

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

复溶计算器