计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
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产品简介:
本试剂盒适用于从石蜡包埋组织中有效纯化基因组DNA及总RNA。本品使用专门 优化脱蜡剂和裂解液，释放组织切片样本中的DNA、RNA，不涉及有机试剂二甲苯， 无需过夜操作；消化后的样品在较高的温度孵育后，去除由交联造成的抑制作用，有 效提高核酸的产量和纯度；优化的缓冲系统使裂解液中的核酸可特异结合到吸附膜 上，抑制剂通过两步漂洗步骤有效去除，最后使用低盐缓冲液或水洗脱，即可获得高 纯度DNA、RNA。同时配置高效微量吸附柱，洗脱体积可低至20μl。经过纯化的 DNA、RNA可以直接用于PCR、Real-time PCR、SNP基因分型、STR基因分型、二 代测序、药物基因组学研究和印迹分析等实验。
自备试剂：
无水乙醇
实验前准备及重要注意事项
1.获得样品后，要尽快将样品固定，固定时间以14-24小时为宜，时间过长易导致 DNA、RNA断裂，影响下游实验。如果甲醛固定时间过长或样本存放时间过久 （＞1年）则易导致DNA完整性受损，无法扩出长片段。
2.确保包埋前的样品彻底脱水，残留的福尔马林会抑制Proteinase K的作用。
3.向Proteinase K中加入1.25 ml Proteinase K Storage Buffer使其溶解，-20℃保存。 配制好的Proteinase K勿长时间室温放置，以免影响其活性。
4.第一次使用前应按试剂瓶标签说明先在Buffer RW2、Buffer GW1和Buffer GW2中 加入无水乙醇。
5.使用前请检查Buffer GTL、Buffer GL和Buffer DS是否出现结晶或者沉淀，如有结 晶或者沉淀，请将Buffer GTL、Buffer GL和Buffer DS于37℃水浴重新溶解。
6.实验开始前将水浴锅或恒温混匀仪预热至56℃。
7.请使用常温离心机，或将离心机温度设置为25℃，若温度低于15℃可能会导致吸 附柱堵塞。
8.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。
操作步骤
石蜡包埋样本
1.将组织块多余的石蜡修剪掉，露出组织后切成5-10 µm的薄片。
2.取约1×1 cm2 的切片（共约1-5片切片）置于离心管（自备）中，加入500 μl Buffer DS， 涡旋震荡10秒。短暂离心将样本收集到管底。56°C孵育3分钟，水浴锅中取出后静 置，降至室温后进行下一步操作。 注意：如果样品表面暴露在空气中，最初的2-3片弃掉不用。
3.12,000 rpm离心2分钟，小心彻底吸弃上清液，不要吸到沉淀。可用小枪头（10μl）小 心去除残留的脱蜡液。
4.上述管中加入180 μl Buffer GTL和20 μl Proteinase K，涡旋震荡混匀。
5.56℃孵育15分钟，然后冰上放置3分钟。室温，12,000 rpm离心15分钟。
6.转移上清液至新的1.5 ml离心管用于RNA提取，注意不要吸到未消化组织。将沉淀 用于DNA提取。
RNA提取
7.把第6步得到的上清液，80℃孵育15分钟。
8.加入320 μl Buffer GL，涡旋震荡混匀后加入720 μl无水乙醇，立即涡旋震荡混匀。
9.所得的溶液全部加入到已装入收集管的吸附柱（Spin Columns RS）中，一次不能 加完溶液，可分多次转入。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：若吸附柱堵塞，可能是样本量过多，应考虑将起始切片数量减少为1-2片。
可选步骤：
a. 向吸附柱中加入350 μl Buffer RW1，12,000 rpm离心1分钟，弃废液，将吸附柱重 新放回收集管中。
b. 配制DNase I 混合液：取52 μl RNase-Free Water，向其中加入8 μl 10×Reaction Buffer和20 μl DNase I（1 U/μl），混匀， 配制成终体积为80 μl的反应液。
c. 向吸附柱中直接加入80 µl DNase I 混合液，20-30℃孵育15分钟。
d. 向吸附柱中加入350 μl Buffer RW1，12,000 rpm离心1分钟，弃废液，将吸附柱重 新放回收集管中。 向吸附柱中加入500 μl Buffer RW2，12,000 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。若需去除基因组DNA，可按照以下步骤进行
11.重复步骤10。再12,000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温5 分钟以彻底晾干。
12. 将吸附柱置于一个新的无RNase离心管中，向吸附柱的中间部位悬空加入20-50 μl RNase-Free Water，室温放置5分钟，12,000 rpm离心1分钟，收集RNA溶液， -80℃保存。
DNA提取
7.取第6步获得的沉淀，加入180 μl Buffer GTL和20 μl Proteinase K至沉淀中。涡旋 15秒重悬沉淀。
8.56℃孵育1小时，直至样品完全溶解。90℃孵育1小时。
9.加入200 μl Buffer GL，涡旋震荡混匀后加入200 μl无水乙醇，涡旋震荡彻底混匀。 短暂离心，使管壁上的溶液收集到管底。
1.将步骤9所得溶液全部加入已装入收集管的吸附柱（Spin Columns DF）中，一次 不能加完溶液，可分多次转入。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：若吸附柱堵塞，可能是样本量过多，应考虑将起始切片数量减少为1-2片。
11.向吸附柱中加入500 μl Buffer GW1，12,000 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。
12.向吸附柱中加入500 μl Buffer GW2，12,000 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：如需进一步提高纯度，可重复步骤12。
13.12,000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温5分钟以彻底晾干。 注意：这一步目的是将吸附柱中残余乙醇去除，乙醇残留会影响后续酶促反应。
14.将吸附柱置于一个新的1.5 ml离心管中，向吸附柱的中间部位悬空加入20-50 μl Buffer EB，室温放置5分钟，12,000 rpm离心1分钟，收集DNA溶液，-20℃保存。
FFPE DNA/RNA Kit
Fixed Tissue DNA/RNA Extraction Kit
Catalog number: F666120 (50 preps)
Storage conditions: DNase I and 10×Reaction Buffer -20℃, Spin Columns DF and Spin Columns RS can be stored at room temperature for 2 months, 2-8℃ for 1 year, the rest of the components are stored at room temperature (15-30℃).
Products Content:
Products Introduction
This kit is suitable for the effective purification of genomic DNA and total RNA from paraffin-embedded tissues, using specially optimized deparaffinizing agents and lysates to release DNA and RNA from tissue section samples, without the use of the organic reagent xylene, and without the need for overnight operation; the digested samples are incubated at higher temperatures to remove inhibitors caused by cross-linking, which can effectively improve nucleic acid yields and purity; and an optimized buffer system allows nucleic acids in the lysate to bind specifically to the adsorbent membrane, and inhibitors are effectively removed by a two-step rinsing procedure. The optimized buffer system enables the nucleic acids in the lysate to bind specifically to the adsorbent membrane, and the inhibitors are effectively removed by a two-step rinsing step, and finally eluted with low-salt buffer or water to obtain high purity DNA and RNA, and at the same time, equipped with a high-efficiency microsorbent column, the volume of the elution can be as low as 20 μl. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, and so on. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, second-generation sequencing, pharmacogenomics research and blot analysis.
Self-contained reagent: anhydrous ethanol
Pre-experiment Preparation and Important Notes
1. After obtaining the sample, fix the sample as soon as possible, the fixation time of 14-24 hours is appropriate, too long a period of time will easily lead to
DNA and RNA breaks, affecting downstream experiments. If the formaldehyde fixation time is too long or the sample is stored for too long
(>1 year) is prone to compromise DNA integrity and failure to amplify long fragments.
2. Ensure that samples are thoroughly dehydrated prior to embedding; residual formalin will inhibit Proteinase K action.
3. Add 1.25 ml of Proteinase K Storage Buffer to Proteinase K to dissolve it, and store at -20℃. Do not leave the prepared Proteinase K at room temperature for a long period of time to avoid affecting its activity.
4. Anhydrous ethanol should be added to Buffer RW2, Buffer GW1 and Buffer GW2 according to the label instructions on the vials before first use.
5. Check Buffer GTL, Buffer GL and Buffer DS for crystallization or precipitation prior to use; if crystallization or precipitation occurs, redissolve Buffer GTL, Buffer GL and Buffer DS in a 37°C water bath.
6. Preheat the water bath or thermostatic mixer to 56°C before starting the experiment.
7. Use an ambient temperature centrifuge or set the centrifuge temperature to 25°C. Temperatures below 15°C may result in clogging of the adsorption column.
8. To prevent RNase contamination, the following should be observed:
1) Use RNase-free plastics and tips to avoid cross-contamination.
(2) Glassware should be dry baked at 180°C for 4 hours before use, plasticware can be soaked in 0.5 M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.
3) RNase-free water should be used to prepare the solution.
(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.
procedure
Paraffin-embedded samples
1. Trim off excess paraffin from the tissue block to expose the tissue and cut into 5-10 µm slices.
2. Place approximately 1 x 1 cm2 slices (1-5 slices in total) in a centrifuge tube (supplied), add 500 μl of Buffer DS and vortex for 10 s. Briefly centrifuge the sample to the bottom of the tube. Centrifuge briefly to collect the sample at the bottom of the tube, incubate at 56°C for 3 minutes, remove from the water bath and allow to cool to room temperature before proceeding.
Note: If the surface of the sample is exposed to air, discard the initial 2-3 slices without using them.
3. Centrifuge at 12,000 rpm for 2 minutes and carefully discard the supernatant thoroughly without aspirating the precipitate. The residual dewaxing solution can be carefully removed with a small tip (10 μl).
4. Add 180 μl of Buffer GTL and 20 μl of Proteinase K to the above tube and mix well with vortexing.
5. Incubate at 56°C for 15 minutes, then place on ice for 3 minutes. Centrifuge at 12,000 rpm for 15 minutes at room temperature.
6. Transfer the supernatant to a new 1.5 ml centrifuge tube for RNA extraction, taking care not to aspirate undigested tissue. Use the precipitate for DNA extraction.
RNA extraction
7. Take the supernatant obtained in step 6 and incubate at 80°C for 15 minutes.
8. Add 320 μl of Buffer GL, mix by vortexing and shaking, then add 720 μl of anhydrous ethanol and mix immediately by vortexing and shaking.
9. Add all of the resulting solution to the Spin Columns RS in the collection tube; if the solution cannot be added all at once, it may be transferred in several passes. centrifuge the column at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and place the column back into the collection tube. Note: If the columns are clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.
Optional step: If genomic DNA is to be removed, the following steps can be followed
a. Add 350 μl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and place the column back into the collection tube.
b. Preparation of DNase I mixture: Take 52 μl of RNase-Free Water and add 8 μl of 10×Reaction to it.
Buffer and 20 μl DNase I (1 U/μl), mix well, and prepare a final volume of 80 μl of reaction solution.
c. Add 80 µl of DNase I Mix directly to the adsorption column and incubate at 20-30°C for 15 minutes.
d. Add 350 μl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.
Add 500 μl of Buffer RW2 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.
11. Repeat step 10. centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the column at room temperature for 5 minutes.
minutes to dry thoroughly.
12. Place the column in a new RNase-free centrifuge tube and add 20-50 μl to the center of the column.
RNase-Free Water, left at room temperature for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and collected RNA solution, the
-80°C for storage.
DNA extraction
7. Take the precipitate obtained in step 6 and add 180 μl Buffer GTL and 20 μl Proteinase K to the precipitate. Vortex
Resuspend the precipitate for 15 seconds.
8. Incubate at 56°C for 1 hour until the sample is completely dissolved. 90°C for 1 hour.
Add 200 μl Buffer GL, vortex and shake to mix and then add 200 μl anhydrous ethanol, vortex and shake to mix thoroughly. Centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.
Add all of the solution from step 9 to the Spin Columns DF in the collection tube, or transfer the solution in several passes. centrifuge at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and return the column to the 10. collection tube.
Note: If the adsorption column is clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.
11. Add 500 μl of Buffer GW1 to the adsorbent column and centrifuge at 12,000 rpm for 1 minute. Pour off the waste liquid from the collection tube and put the column back into the collection tube.
12. Add 500 μl of Buffer GW2 to the adsorbent column and centrifuge at 12,000 rpm for 1 minute. Pour off the waste liquid from the collection tube and place the column back into the collection tube.
Note: Step 12 may be repeated if further purity is required.
13. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for 5 minutes to dry thoroughly.
Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue will affect the subsequent enzymatic reaction. 14. Place the column in a new 1.5 ml centrifuge tube, add 20-50 μl Buffer EB to the center of the column, leave at room temperature for 5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.
规格
英文名称
FFPE DNA/RNA Kit
储存与运输
储存条件
-20°C储存
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
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溶液计算器
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