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
2-8°C储存,室温。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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
产品内容
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产品简介
本试剂盒适用于从福尔马林固定、石蜡包埋组织中有效纯化基因组DNA。本品使用专门优化脱蜡剂和裂解液，释放福尔马林固定或组织切片样本中的DNA，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，去除游离DNA的福尔马林交联，有效提高DNA的产量和纯度；优化的缓冲系统使裂解液中的DNA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。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可以直接用于PCR、Real-time PCR、SNP 基因分型、STR基因分型、二代测序和药物基因组学研究等。
从福尔马林固定、石蜡包埋样本中分离的DNA分子量通常低于新鲜或冷冻样本中的DNA。DNA片段化的程度取决于样本类型、储存时间以及固定的条件。
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，要尽快将样品在4%-10%的福尔马林中固定，固定时间以14-24小时为宜，时间过长易导致基因组断裂，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. 第一次使用前应按试剂瓶标签说明先在Buffer GW1和Buffer GW2中加入无水乙醇。
4. 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56℃水浴重新溶解。
5. 实验开始前将水浴锅或恒温混匀仪预热至56℃，离心机保持25℃。
6. 如果下游实验需要降低低频发生的 C>T | G>A 转换（人为突变），将假阳性风险降至最低，可以在90℃孵育1小时后加入7 μL UNG（1U/uL）。
操作步骤
1. 样本处理：
1a. 石蜡包埋样本：用手术刀将组织块中多余的石蜡修剪掉，露出组织后切成5-10 µm 的薄片。取约1×1 cm2的切片（共约4-5片切片）置于离心管（自备）中，加入160 μL Buffer DS，涡旋震荡10秒，再加入180 μL Buffer GTL和20μL Proteinase K，涡旋震荡10秒。12000 rpm，25℃ 离心1分钟。
注意：1 ) 如果样品表面已经暴露在空气中，请将接触空气的2-3片弃掉不用。
2）DS低于18℃会凝固，如果凝固不影响下面的实验。
1b. 福尔马林等固定液中的样本：取约20 mg的样本，切成小块，置于离心管中，加入
500 μL 10mM PBS（PH7.4），涡旋振荡，12000 rpm 离心1分钟，弃上清，重复3 次。加入180 μL Buffer GTL， 20 μL Proteinase K，涡旋震荡混匀。
2. 56℃孵育1小时，直至样品完全溶解。90℃孵育1小时。12000 rpm，25℃ 离心1分钟，用移液器沿管壁小心吸取下层水相（约180μL）于新离心管中，尽量避免吸入管底沉淀和上层蜡液。
注意：1）56℃孵育后的样品可置于室温，直至水浴锅或干浴锅温度达到90℃后再把样品置于90℃孵育。
2）可选步骤：加入7 μL UNG（1U/μL），50℃，5 min ，不震荡。此步骤的目的是降低低频发生的 C>T | G>A转换（人为突变），同时有效保留真实发生的突变，从而使假阳性风险降至最低。
3. 可选步骤：如需除去RNA，可将样品温度降到室温后，加入2 μL浓度为100 mg/mL的RNase A 溶液，震荡混匀，室温放置2分钟。
4. 加入20 μL Proteinase K，65℃，450 rpm孵育15 min 。
5. 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。
注意：1）加入Buffer GL和无水乙醇后要立即充分混匀。
2） 加入Buffer GL和无水乙醇后可能会产生白色沉淀，不会影响后续实验。
3） 如果需要对多个样品进行操作，可以将Buffer GL和无水乙醇事先混匀后加样。
6. 将步骤5所得溶液全部加入已装入收集管的吸附柱（Spin Columns DF）中， 25℃ ，12000 rpm 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。
7. 向吸附柱中加入500 μL Buffer GW1（使用前检查是否已加入无水乙醇），12000 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。
8. 
向吸附柱中加入500 μL Buffer GW2（使用前检查是否已加入无水乙醇），12000 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DNA纯度，可重复步骤8。
9. 12000 rpm离心2分钟，倒掉收集管中废液。将吸附柱置于室温数分钟以彻底晾干。
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。
10. 将吸附柱置于一个新的1.5 mL收集管中，向吸附柱的中间部位悬空加入20-100 μLBuffer TE或灭菌水，室温放置2-5分钟，12000 rpm离心1分钟，收集DNA溶液，-20℃保存DNA。
注意：1）洗脱液的pH值对洗脱效率有很大影响，若用水做洗脱液应保证其pH值在7.0-8.5，pH 值低于7.0时洗脱效率不高。
2）如果要提高DNA的终浓度，可以将步骤10所得的DNA洗脱液重新加至吸附膜上，室温放置2分 钟，12000 rpm离心1分钟。
Product content
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Product Introduction
This kit is suitable for the effective purification of genomic DNA from formalin-fixed, paraffin-embedded tissues.The product uses specially optimized dewaxing agent and lysis solution to release DNA from formalin-fixed or tissue sectioned samples, which does not involve the organic reagent xylene and does not need to be operated overnight; the digested samples are incubated at higher temperatures to remove formalin cross-linking of the free DNA, which can effectively improve the yield and purity of DNA; the optimized buffer system allows the inhibitors in the lysis solution to be specifically bound to the adsorbent membrane, which can be effectively removed by a two-step rinsing step. The optimized buffer system enables the DNA in the lysate to specifically bind to the adsorbent membrane, and the inhibitor is effectively removed by a two-step rinsing step, and finally eluted with low-salt buffer or water to obtain high-purity DNA.Meanwhile, configured with a high-efficiency microsorbent column, the elution volume can be as low as 20 μL.The purified DNA can be directly used for PCR, Real-time PCR, SNP Genotyping, STR genotyping, second-generation sequencing and pharmacogenomics research.
The molecular weight of DNA isolated from formalin-fixed, paraffin-embedded samples is usually lower than that of DNA from fresh or frozen samples.The degree of DNA fragmentation depends on the type of sample, the duration of storage, and the conditions of fixation.
Self-contained reagent: anhydrous ethanol
Pre-experiment Preparation and Important Notes
1. After obtaining the sample, fix the sample in 4%-10% formalin as soon as possible, the fixation time should be 14-24 hours, too long a period of time will easily lead to genome breakage, affecting the downstream experiments. If the formaldehyde fixation time is too long or the sample has been stored for too long (> 1 year), it will easily lead to DNA integrity damage and unable to amplify long fragments.
2. Ensure that the sample is thoroughly dehydrated before embedding; residual formalin will inhibit Proteinase K.
3. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.
4. Before use, please check Buffer GTL, Buffer GL and Buffer DS for any crystallization or precipitation. If there is any crystallization or precipitation, please re-dissolve Buffer GTL, Buffer GL and Buffer DS at 56℃ in a water bath.
5. Preheat the water bath or thermostatic mixer to 56°C and keep the centrifuge at 25°C before starting the experiment.
6. If downstream experiments are needed to reduce the low frequency of C>T|G>A transitions (artificial mutations) that occur to minimize the risk of false positives, 7 μL of UNG (1 U/uL) can be added after 1 hour of incubation at 90°C.
Operation steps
1. Sample processing:
1a. Paraffin-embedded samples: Trim off excess paraffin from the tissue block with a scalpel to expose the tissue and then cut into 5-10µm slices. Take about 1×1cm2 slices (about 4-5 slices in total) and place them in a centrifuge tube (provided), add 160μL Buffer DS, vortex and shake for 10 seconds, then add 180μL Buffer GTL and 20μL Proteinase K, vortex and shake for 10 seconds. centrifuge the samples at 12,000rpm for 1 minute at 25℃.
Note: 1) If the surface of the sample has been exposed to air, discard the 2-3 pieces that have been exposed to air and do not use them.
2) DS will solidify below 18°C, and if it does it does not affect the following experiments.
1b. Sample in formalin and other fixative: take about 20mg of sample, cut it into small pieces, place it in a centrifuge tube, add 500μL of 10mM PBS (PH7.4), vortex shaking, centrifuge at 12,000rpm for 1minute, discard the supernatant, and repeat 3 times. Add 180 μL Buffer GTL, 20 μL Proteinase K, vortex shaking to mix.
2.56°C for 1 hour until the sample is completely dissolved. incubate at 90°C for 1 hour. centrifuge at 12,000 rpm, 25°C for 1 minute, and carefully pipette the lower aqueous phase (~180 μL) along the wall of the tube into a new centrifuge tube, trying to avoid aspirating the bottom precipitate and the upper layer of the wax solution.
Note: 1) Samples can be left at room temperature after incubation at 56°C until the temperature of the water or dry bath reaches 90°C before placing the samples at 90°C
Incubation.
2) Optional step: add 7μL UNG (1U/μL), 50°C, 5min, no shaking. The purpose of this step is to minimize the risk of false positives by reducing the low-frequency occurrence of C>T|G>A transitions (artificial mutations) while effectively retaining the true occurrence of mutations.
3. Optional step: If you need to remove RNA, you can lower the temperature of the sample to room temperature, then add 2μL of RNase A solution at a concentration of 100mg/mL, shake and mix well, and leave it at room temperature for 2 minutes.
4. Add 20μL Proteinase K and incubate at 65℃, 450rpm for 15min.
5. Add 200 μL of Buffer GL, mix well by vortexing and shaking, then add 200 μL of anhydrous ethanol and mix thoroughly by vortexing and shaking. Centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.
Note: 1) Mix well immediately after adding Buffer GL and anhydrous ethanol.
2) The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.
3) If more than one sample needs to be manipulated, the Buffer GL and anhydrous ethanol can be pre-mixed and spiked.
6. Add all the solution obtained in step 5 to the adsorption columns (Spin Columns DF) that have been loaded into the collection tube, centrifuge at 25℃, 12000rpm for 2 minutes, pour out the waste liquid in the collection tube, and put the adsorption columns back into the collection tube.
7. Add 500μL of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
8. Add 500μL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12000rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
Note: Step 8 can be repeated if further DNA purity is required.
9.12 Centrifuge at 2000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.
Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions.
10. Place the adsorption column in a new 1.5 mL collection tube, add 20-100 μL of Buffer TE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, and collect the DNA solution.
-20°C to preserve DNA.
Note: 1) The pH value of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH value is 7.0-8.5, the pH value is lower than 7.0 when the elution efficiency is not high.
2) If the final concentration of DNA is to be increased, the DNA eluate obtained in step 10 can be re-spiked onto the adsorbent membrane and left at room temperature for 2 minutes and centrifuged at 12,000 rpm for 1 minute.
规格
英文名称
NuClean FFPE DNA Kit
储存与运输
储存条件
2-8°C储存,室温
运输条件
低温运输
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溶液计算器
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