QIAGEN-DNeasy mericon food handbook
QIAamp
本操作手册适用于离心法从140ul 血清、血浆、尿液、细胞培养上清液、无细胞体液、拭子浸液中纯化病毒RNA 。
如需配合QIAcube 核酸纯化工作站实现自动化病毒RNA 纯化，请参阅QIAcbue 操作说明。
如处理更大量的样本（可多至560ul ），按照标准操作流程增加适当比例的裂解液以及载样次数即可。
针对一些病毒含量很低的样本，可参照附录中样本浓缩操作步骤对样本进行浓缩。
注意事项注意事项：：1、 所有步骤所有步骤均均于室温下室温下操作操作(15–25°C)。
2、 样本收集和离心后，血浆（未经处理，或用肝素之外的抗凝剂处理）或血清可在2–8°C 下储存至多6个小时。
如需长期保存，推荐分装后在–20°C 或 –80°C 保存。
冷冻血浆或血清样本不可反复冻融样本不可反复冻融样本不可反复冻融。
反复冻融将会促使蛋白变性沉淀，因而病毒含量降低，导致纯化得到的病毒RNA 的量减少。
并且，反复冻融形成的冷凝蛋白将有可能导致QIAamp 膜堵塞。
冷凝蛋白可通过 6800 x g 下快速离心3 分钟去除，小心吸取上清后立即进行下一步操作。
这一步骤不会影响病毒的含量。
3、 本操作步骤不适用于RNA 和DNA 分离，样本中存在的样本中存在的DNA 和RNA 会被同时纯化出来会被同时纯化出来。
如需完全去除DNA 的影响，推荐使用无细胞体液作为病毒RNA 纯化的起始样本。
含有细胞的样本，例如脑脊液、骨髓、尿液、以及大部分拭子，首先需要过滤，或者1500 x g 离心10分钟以上取上清使用。
如DNA 和RNA 已经一并完成纯化，可将洗脱液经过无RNA 酶的DNA 酶消化处理，接着热处理将DNA 酶灭活。
4、 可以纯化所有大于大于200nt 的RNA 分子分子，小分子RNA 将不能得到高效率的纯化。
本操作手册仅限内部学习使用！如有任何疑问，请联系：胡川（189****3075）准备事项准备事项：：1、 所有样本所有样本平衡至室温平衡至室温2、 Buffer AVE 平衡至室温3、 每个样本每个样本提供提供4个2ml 收集管收集管，，根据不同需要适当自备根据不同需要适当自备无无RNA 酶超净2ml 收集管/1.5ml 离心管4、 按照按照下面的配方准备下面的配方准备AW1 和AW2 BufferBuffer AW1以浓缩液的形式提供。
Rotor-GeneQ实时荧光定量PCR分析仪-Qiagen
Rotor-Gene Q 可以带给您：离心式的设计带给您出色的温度和光学表现■宽广的光谱范围，从紫外到红外波长■坚固耐用的设计，确保低维护和高度方便性■HRM遗传分析功能与定量扩增相结合■配合QIAGEN 试剂盒，在多种应用方向都有出色表现■广泛的应用领域Rotor-Gene Q 与QIAGEN 试剂盒相结合，实现所有定量PCR 的应用，以及高分辨率熔解（HRM®）分析：基因表达分析■■基因分型病原体检测■■基因扫描DNA 甲基化分析■■ miRNA研究物种、品种鉴定■■ HLA 配型分析详细应用参见第8页。
访问/PCR-applications并浏览Rotor-Gene Q模拟世界，体验超过20个仪器与软件的动画演示。
Rotor-Gene Q ——您实验成功的保障实时定量PCR 是一门精确的技术，它对仪器、试剂和软件都有极高的要求。
高度的温度和光学均一性、短时间内的温度平衡，以及快速的升降温对于精确和快速的定量分析都是极其重要的。
同样，检测的灵敏度、速度和特异性也高度依赖于DNA 聚合酶和反应组份。
QIAGEN公司的实时定量PCR分析仪Rotor-Gene Q将多种优化的设计相结合，为您精密的研究需求提供出色而可靠的实验结果。
配合QIAGEN 专为定量PCR 而优化的试剂盒，Rotor-Gene Q 可以让您直接、高效的将其应用于广泛的研究领域。
离心式的设计，出色的表现Rotor-Gene Q 独特的离心转子式设计让她成为在精确和通用性方面都非常出色的实时定量PCR分析仪（图1）。
每个PCR 管在反应仓里匀速旋转，空气不断高速流动，所有样品在快速的升降温过程中始终处于高度一致的温度下。
信号检测也具有同样的均一性。
当管子对准检测光路时，样品就被激发，荧光信号快速通过单一的短光路被PMT（光电倍增管）收集。
温度和光学上的均一性保证了定量PCR 分析的灵敏、精确和快速（图2）。
并且这种离心式的设计还消除了管和管之间的差异及边缘效应，而传统板式仪器由于板块内部温度的差异和复杂的光路系统无法具备Rotor-Gene Q 的这些出色性能。
细胞RNA提取说明书 RNeasy Plus Mini Handbook
September 2010RNeasy® Plus Mini HandbookFor purification of total RNA from animalcells and easy-to-lyse animal tissues usinggDNA Eliminator columnsQIAGEN Sample and Assay TechnologiesQIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result.QIAGEN sets standards in:Purification of DNA, RNA, and proteinsNucleic acid and protein assaysmicroRNA research and RNAiAutomation of sample and assay technologiesOur mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit .ContentsKit Contents 4 Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Quality Control 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Determining the amount of starting material 12 Handling and storing starting material 15 Disrupting and homogenizing starting material 15 ProtocolsPurification of Total RNA from Animal Cells 17 Purification of Total RNA from Animal Tissues 23 Troubleshooting Guide 30 Appendix A: General Remarks on Handling RNA 34 Appendix B: Storage, Quantification, and Determination of Qualityof RNA 36 Appendix C: Protocol for Formaldehyde Agarose Gel Electrophoresis 39 Appendix D: Purification of Total RNA Containing Small RNAsfrom Cells 41 Appendix E: Separate Purification of Small RNA (Containing miRNA)and Larger RNA from Animal Cells Using the RNeasy Plus Mini Kitand RNeasy MinElute Cleanup Kit 43 References 46 Ordering Information 47Kit ContentsRNeasy Plus Mini Kit (50) (250)Catalog no. 74134 74136Number of preps 50 250gDNA Eliminator Mini Spin Columns (uncolored)50 250(each in a 2 ml Collection Tube)50 250RNeasy Mini Spin Columns (pink)(each in a 2 ml Collection Tube)Collection Tubes (1.5 ml) 50 250Collection Tubes (2 ml) 50 250Buffer RLT Plus* 45 ml 220 mlBuffer RW1* 45 ml 220 mlBuffer RPE† (concentrate) 11 ml 55 mlRNase-Free Water 10 ml 50 mlHandbook 11* Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6for safety information.† Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on thebottle to obtain a working solution.StorageThe RNeasy Plus Mini Kit should be stored dry at room temperature (15–25ºC)and is stable for at least 9 months under these conditions.Product Use LimitationsThe RNeasy Plus Mini Kit is intended for molecular biology applications. Thisproduct is not intended for the diagnosis, prevention, or treatment of a disease.All due care and attention should be exercised in the handling of the product.We recommend all users of QIAGEN® products to adhere to the NIH guidelinesthat have been developed for recombinant DNA experiments, or to otherapplicable guidelines.Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner describedin our product literature. The purchaser must determine the suitability of theproduct for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge orrefund the purchase price. We reserve the right to change, alter, or modify anyproduct to enhance its performance and design. If a QIAGEN product does notmeet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product — as you wish. Separate conditions apply to QIAGEN scientific instruments, service products, and to products shipped on dry ice. Please inquire for more information.A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see back cover or visit ).Technical AssistanceAt QIAGEN, we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products. If you have any questions or experience any difficulties regarding the RNeasy Plus Mini Kit or QIAGEN products in general, please do not hesitate to contact us.QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques.For technical assistance and more information, please see our Technical Support Center at /Support or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit ).Safety InformationWhen working with chemicals, always wear a suitable lab coat, disposablegloves, and protective goggles. For more information, please consult theappropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at /ts/msds.asp whereyou can find, view, and print the MSDS for each QIAGEN kit and kitcomponent.CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.Buffer RLT Plus contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate. This chemical can form highly reactive compounds when combined with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent and water. If the spilt liquid contains potentially infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite. The following risk and safety phrases apply to the components of the RNeasy Plus Mini Kit.Buffer RLT PlusContains guanidine thiocyanate: harmful. Risk and safety phrases:*R20/21/22-32, S13-26-36-46Buffer RW1Contains ethanol: flammable. Risk phrase:* R1024-hour emergency informationEmergency medical information in English, French, and German can be obtained 24 hours a day from:Poison Information Center Mainz, GermanyTel: +49-6131-19240* R10: Flammable; R20/21/22: Harmful by inhalation, in contact with skin, and if swallowed; R32: Contact with acids liberates very toxic gas; S13: Keep away from food, drink and animal feeding stuffs; S26: In case of contact with eyes, rinse immediately with plenty of water and seek medical advice; S36: Wear suitable protective clothing; S46: If swallowed, seek medical advice immediately and show the container or label.Quality ControlIn accordance with QIAGEN’s ISO-certified Quality Management System, each lot of RNeasy Plus Mini Kit is tested against predetermined specifications to ensure consistent product quality.IntroductionThe RNeasy Plus Mini Kit is designed to purify RNA from small amounts ofanimal cells or tissues. The kit is compatible with a wide range of cultured cellsand with easy-to-lyse tissues. Genomic DNA contamination is effectively removed using a specially designed gDNA Eliminator spin column. The purifiedRNA is ready to use and is ideally suited for downstream applications that aresensitive to low amounts of DNA contamination, such as quantitative, real-timeRT-PCR.* The purified RNA can also be used in other applications, including:RT-PCRDifferential displaycDNA synthesisNorthern, dot, and slot blot analysesPrimer extensionPoly A+ RNA selectionRNase/S1 nuclease protectionMicroarraysThe RNeasy Plus Mini Kit allows the parallel processing of multiple samples inless than 25 minutes. Time-consuming and tedious methods such as CsCl step-gradient ultracentrifugation and alcohol precipitation steps, or methods involving the use of toxic substances such as phenol and/or chloroform, are replaced by the RNeasy Plus procedure.Principle and procedureThe RNeasy Plus procedure integrates QIAGEN’s patented technology for selective removal of double-stranded DNA with well-established RNeasy technology. Efficient purification of high-quality RNA is guaranteed, without the need for additional DNase digestion.Biological samples are first lysed and homogenized in a highly denaturing guanidine-isothiocyanate–containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. The lysate is then passed through a gDNA Eliminator spin column. This column, in combination with the optimized high-salt buffer, allows efficient removal of genomic DNA.Ethanol is added to the flow-through to provide appropriate binding conditions for RNA, and the sample is then applied to an RNeasy spin column, where total* Visit /geneXpression for information on standardized solutions for gene expression analysis, including QuantiTect® Kits and Assays for quantitative, real-time RT-PCR.RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in 30 μl, or more, of water.With the RNeasy Plus procedure, all RNA molecules longer than 200 nucleotides are isolated. The procedure provides an enrichment for mRNA, since most RNAs <200 nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA) are selectively excluded. The size distribution of the purified RNA is comparable to that obtained by centrifugation through a CsCl cushion, where small RNAs do not sediment efficiently.In this handbook, different protocols are provided for different starting materials. The protocols differ primarily in the lysis and homogenization of the sample. Once the sample is applied to the gDNA Eliminator spin column, the protocols are similar (see flowchart, next page).RNeasy Plus Procedure CellsortissueGenomic DNATotal RNAEluted RNA Lyse and homogenize Remove genomic DNA Add ethanolBind total RNAWashEluteEquipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.For all protocols14.3 M β-mercaptoethanol (β-ME) (commercially available solutions areusually 14.3 M)Sterile, RNase-free pipet tipsMicrocentrifuge (with rotor for 2 ml tubes)96–100% ethanol*70% ethanol* in waterDisposable glovesFor tissue samples: RNA later® RNA Stabilization Reagent (see ordering information, page 47) or liquid nitrogenEquipment for sample disruption and homogenization (see pages 15–16).Depending on the method chosen, one or more of the following arerequired:Trypsin and PBSQIAshredder homogenizer (see ordering information, page 47)Blunt-ended needle and syringeMortar and pestleTissueLyser II or TissueLyser LT (see ordering information, page 48) TissueRuptor® homogenizer (see ordering information, page 48)* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.Important NotesDetermining the amount of starting materialIt is essential to use the correct amount of starting material in order to obtain optimal RNA yield and purity. The maximum amount that can be used is limited by:The type of sample and its DNA and RNA contentThe volume of Buffer RLT Plus required for efficient lysis and the maximum loading volume of the RNeasy spin columnThe DNA removal capacity of the gDNA Eliminator spin columnThe RNA binding capacity of the RNeasy spin columnWhen processing samples containing high amounts of DNA or RNA, less than the maximum amount of starting material shown in Table 1 should be used, so that the DNA removal capacity of the gDNA Eliminator spin column and the RNA binding capacity of the RNeasy spin column are not exceeded.When processing samples containing average or low amounts of RNA, the maximum amount of starting material shown in Table 1 can be used. However, even though the RNA binding capacity of the RNeasy spin column is not reached, the maximum amount of starting material must not be exceeded. Otherwise, lysis will be incomplete and cellular debris may interfere with the binding of RNA to the RNeasy spin column membrane, resulting in lower RNA yield and purity.More information on using the correct amount of starting material is given in each protocol. Table 2 shows expected RNA yields from various cells and tissues.Note: If the DNA removal capacity of the gDNA Eliminator spin column is exceeded, the purified RNA will be contaminated with DNA. Although the gDNA Eliminator spin column can bind more than 100 μg DNA, we recommend using samples containing less than 20 μg DNA to ensure removal of virtually all genomic DNA. If the binding capacity of the RNeasy spin column is exceeded, RNA yields will not be consistent and may be reduced. If lysis of the starting material is incomplete, RNA yields will be lower than expected, even if the binding capacity of the RNeasy spin column is not exceeded.Table 1. RNeasy Mini spin column specificationsMaximum binding capacity 100 μg RNA Maximum loading volume 700 μlRNA size distribution RNA >200 nucleotides Minimum elution volume 30 μl Maximum amount of starting materialAnimalcells 1 x 107 cellsAnimal tissues 30 mgTable 2. Yields of total RNA with the RNeasy Plus Mini KitCell cultures (1 x 106 cells) Average yield oftotal RNA* (μg)Mouse/rattissues (10 mg)Average yield oftotal RNA* (μg)NIH/3T3 10 Embryo (13 day) 25HeLa 15 Brain 5–10COS-7 35Heart 4–8 LMH 12 Kidney 20–30Huh 15Liver 40–60Lung 10–20* Amounts can vary due to factors such as species, developmental stage, and growthconditions. Since the RNeasy procedure enriches for mRNA and other RNA species >200nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15–20% of total cellular RNA.Counting cells or weighing tissue is the most accurate way to quantitate theamount of starting material. However, the following may be used as a guide.Animal cellsThe number of HeLa cells obtained in various culture vessels after confluentgrowth is given in Table 3.Table 3. Growth area and number of HeLa cells in various culturevesselsCell-culture vessel Growth area (cm3)* Number of cells†Multiwell plates96-well 0.32–0.6 4–5104x48-well 1 1 x 10524-well 2 2.5 x 10512-well 4 5 x 1056-well 9.5 1 x 106Dishes 35 mm 8 1 x 10660mm 21 2.5 x 106100 mm 56 7 x 106145–150mm 145 2 x 107Flasks 40–50 ml 25 3 x 106250–300ml 75 1 x 107650–750 ml 162–175 2 x 107* Per well, if multiwell plates are used; varies slightly depending on the supplier.† Cell numbers are given for HeLa cells (approximate length = 15 μm), assuming confluentgrowth. Cell numbers will vary for different kinds of animal cells, which vary in length from10 to 30 μm.Animal tissuesA 3 mm cube (27 mm3) of most animal tissues weighs 30–35 mg.Handling and storing starting materialRNA in harvested tissue is not protected until the sample is treated with RNA laterRNA Stabilization Reagent, flash-frozen, or disrupted and homogenized in thepresence of RNase-inhibiting or denaturing reagents. Otherwise, unwantedchanges in the gene expression profile will occur. It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at –70ºC,or immediately immersed in RNA later RNA Stabilization Reagent.The procedures for tissue harvesting and RNA protection should be carried out as quickly as possible. Frozen tissue samples should not be allowed to thaw during handling or weighing. After disruption and homogenization in Buffer RLT Plus (lysis buffer), samples can be stored at –70ºC for months.Disrupting and homogenizing starting materialEfficient disruption and homogenization of the starting material is an absolute requirement for all total RNA purification procedures. Disruption and homogenization are 2 distinct steps:Disruption: Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the RNA contained in thesample. Incomplete disruption results in significantly reduced RNA yields. Homogenization: Homogenization is necessary to reduce the viscosity of the lysates produced by disruption. Homogenization shears high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column membrane and therefore significantly reduced RNA yields.Disruption and homogenization of tissue samples can be carried out rapidly and efficiently using either the TissueRuptor (for processing samples individually) or a TissueLyser system (for processing multiple samples simultaneously). Disruption and homogenization with TissueRuptor and TissueLyser systems generally results in higher RNA yields than with other methods.Disruption and homogenization using the TissueRuptorThe TissueRuptor is a rotor–stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15–90 seconds, depending on the toughness and size of the sample. The blade of the TissueRuptor disposable probe rotates at a very high speed, causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing. For guidelines on using the TissueRuptor, refer to the TissueRuptor Handbook. For other rotor–stator homogenizers, refer to suppliers’ guidelines.Disruption and homogenization using TissueLyser systemsIn bead-milling, tissues can be disrupted by rapid agitation in the presence ofbeads and lysis buffer. Disruption and simultaneous homogenization occur bythe shearing and crushing action of the beads as they collide with the cells. Twobead mills are available from QIAGEN: the TissueLyser LT for low- to medium-throughput disruption, and the TissueLyser II for medium- to high-throughputdisruption.The TissueLyser LT disrupts and homogenizes up to 12 samples at the same time. The instrument needs to be used in combination with the TissueLyser LT Adapter, which holds 12 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm or 7 mm mean diameter. For guidelines on using the TissueLyser LT, refer to the TissueLyser LT Handbook.The TissueLyser II disrupts and homogenizes up to 48 tissue samplessimultaneously when used in combination with the TissueLyser Adapter Set2 x 24, which holds 48 x 2 ml microcentrifuge tubes containing stainless steelbeads of 5 mm mean diameter. For guidelines on using the TissueLyser II, refer to the TissueLyser Handbook. If using other bead mills for sample disruption and homogenization, refer to suppliers’ guidelines.Note: Tungsten carbide beads react with QIAzol Lysis Reagent and must not be used to disrupt and homogenize tissues.The TissueLyser II can also disrupt and homogenize up to 192 tissue samplessimultaneously when used in combination with the TissueLyser Adapter Set2 x 96, which holds 192 x 1.2 ml microtubes containing stainless steel beads of 5 mm mean diameter. In this case, we recommend using the RNeasy 96 Universal Tissue Kit, which provides high-throughput RNA purification from all types of tissue in 96-well format. For ordering information, see page 48.Protocol: Purification of Total RNA from Animal CellsDetermining the correct amount of starting materialIt is essential to use the correct amount of starting material in order to obtain optimal RNA yield and purity. The minimum amount is generally 100 cells, while the maximum amount depends on:The RNA content of the cell typeThe DNA removal capacity of the gDNA Eliminator spin columnThe RNA binding capacity of the RNeasy spin column (100 μg RNA)The volume of Buffer RLT Plus required for efficient lysis (the maximum volume of Buffer RLT Plus that can be used limits the maximum amount of starting material to 1 x 107 cells)RNA content can vary greatly between cell types. The following examples illustrate how to determine the maximum amount of starting material:COS cells have high RNA content (approximately 35 μg RNA per 106 cells).Do not use more than 3 x 106 cells, otherwise the RNA binding capacity of the RNeasy spin column will be exceeded.HeLa cells have average RNA content (approximately 15 μg RNA per 106 cells). Do not use more than 7 x 106 cells, otherwise the RNA bindingcapacity of the RNeasy spin column will be exceeded.NIH/3T3 cells have low RNA content (approximately 10 μg RNA per 106 cells). The maximum amount of starting material (1 x 107 cells) can beused.If processing a cell type not listed in Table 2 (page 13) and if there is no information about its RNA content, we recommend starting with no more than 3–4 x 106 cells. Depending on RNA yield and purity, it may be possible to increase the cell number in subsequent preparations.Do not overload the gDNA Eliminator spin column, as this will lead to copurification of DNA with RNA. Do not overload the RNeasy spin column, as this will significantly reduce RNA yield and purity.As a guide, Table 3 (page 14) shows the expected numbers of HeLa cells in different cell-culture vessels.Important points before startingIf using the RNeasy Plus Mini Kit for the first time, read “Important Notes”(page 12).If preparing RNA for the first time, read Appendix A (page 34).Cell pellets can be stored at –70ºC for later use or used directly in the procedure. Determine the number of cells before freezing. Frozen cellpellets should be thawed slightly so that they can be dislodged by flickingthe tube in step 2. Homogenized cell lysates from step 3 can be stored at –70ºC for several months. Frozen lysates should be incubated at 37ºC in awater bath until completely thawed and salts are dissolved. Avoidprolonged incubation, which may compromise RNA integrity. If anyinsoluble material is visible, centrifuge for 5 min at 3000–5000 x g.Transfer supernatant to a new RNase-free glass or polypropylene tube, andcontinue with step 4.β-mercaptoethanol (β-ME) must be added to Buffer RLT Plus before use.Add 10 μl β-ME per 1 ml Buffer RLT Plus. Dispense in a fume hood andwear appropriate protective clothing. Buffer RLT Plus is stable at roomtemperature (15–25ºC) for 1 month after addition of β-ME.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%), as indicated on the bottle, to obtain aworking solution.Buffer RLT Plus may form a precipitate during storage. If necessary, redissolve by warming, and then place at room temperature.Buffer RLT Plus and Buffer RW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach. See page 6 for safety information.Perform all steps of the procedure at room temperature. During the procedure, work quickly.Perform all centrifugation steps at 20–25ºC in a standard microcentrifuge.Ensure that the centrifuge does not cool below 20ºC.Procedure1.Harvest cells according to step 1a or 1b.1a. Cells grown in suspension (do not use more than 1 x 107 cells):Determine the number of cells. Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube (notsupplied). Carefully remove all supernatant by aspiration, andproceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilutethe lysate, affecting the conditions for DNA removal and RNA purification.Both effects may reduce RNA quality and yield.1b. Cells grown in a monolayer (do not use more than 1 x 107 cells): Cells grown in a monolayer in cell-culture vessels can be either lyseddirectly in the vessel (up to 10 cm diameter) or trypsinized andcollected as a cell pellet prior to lysis. Cells grown in a monolayer incell-culture flasks should always be trypsinized.To lyse cells directly:Determine the number of cells. Completely aspirate the cell-culturemedium, and proceed immediately to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilutethe lysate, affecting the conditions for DNA removal and RNA purification.Both effects may reduce RNA quality and yield.To trypsinize and collect cells:Determine the number of cells. Aspirate the medium, and wash thecells with PBS. Aspirate the PBS, and add 0.10–0.25% trypsin in PBS.After the cells detach from the dish or flask, add medium (containingserum to inactivate the trypsin), transfer the cells to an RNase-freeglass or polypropylene centrifuge tube (not supplied), and centrifugeat 300 x g for 5 min. Completely aspirate the supernatant, andproceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilutethe lysate, affecting the conditions for DNA removal and RNA purification.Both effects may reduce RNA quality and yield.2.Disrupt the cells by adding Buffer RLT Plus.For pelleted cells, loosen the cell pellet thoroughly by flicking thetube. Add the appropriate volume of Buffer RLT Plus (see Table 5).Vortex or pipet to mix, and proceed to step 3.Note: Incomplete loosening of the cell pellet may lead to inefficient lysisand reduced RNA yields. Ensure that β-ME is added to Buffer RLT Plusbefore use (see “Important points before starting”).Table 5. Volumes of Buffer RLT Plus for lysing pelleted cellsNumber of pelleted cells Volume of Buffer RLT Plusμl<5 x 106 350 5 x 106 – 1 x 107600 μlFor direct lysis of cells grown in a monolayer, add the appropriate volume of Buffer RLT Plus (see Table 6) to the cell-culture dish.Collect the lysate with a rubber policeman. Pipet the lysate into amicrocentrifuge tube (not supplied). Vortex or pipet to mix, andensure that no cell clumps are visible before proceeding to step 3.Note: Ensure that β-ME is added to Buffer RLT Plus before use (see“Important points before starting”).Table 6. Volumes of Buffer RLT Plus for direct cell lysisDish diameter Volume of Buffer RLT Plus*<6 cm 350 μl6–10 cm 600 μl* Regardless of the cell number, use the buffer volumes indicated to completely cover the surface of the dish.3.Homogenize the lysate according to step 3a, 3b, or 3c.See “Disrupting and homogenizing starting material”, page 15, for more details on homogenization. If processing ≤1 x 105 cells, they can behomogenized by vortexing for 1 min. After homogenization, proceed tostep 4.Note: Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the RNeasy spin column. Homogenization with a rotor–stator or QIAshredder homogenizer generally results in higher RNA yields than with a syringe and needle.3a. Pipet the lysate directly into a QIAshredder spin column placed in a2 ml collection tube, and centrifuge for 2 min at maximum speed.Proceed to step 4.3b. Homogenize the lysate for 30 s using the TissueRuptor. Proceed to step 4.3c. Pass the lysate at least 5 times through a 20-gauge needle (0.9 mm diameter) fitted to an RNase-free syringe. Proceed to step 4.4.Transfer the homogenized lysate to a gDNA Eliminator spin columnplaced in a 2 ml collection tube (supplied). Centrifuge for 30 s at≥8000 x g (≥10,000 rpm). Discard the column, and save the flow-through.Note: Make sure that no liquid remains on the column membrane aftercentrifugation. If necessary, repeat the centrifugation until all liquid haspassed through the membrane.。
QIAxcel全自动毛细管核酸分析系统培训手册DNA版
QIAxcel全自动毛细管核酸分析系统培训手册DNA版基因有限公司生命科学组目录一、QIAxcel全自动毛细管核酸分析系统简介二、QIAxcel全自动毛细管核酸分析系统安装和维护三、氮气瓶的安装及使用四、Gel Cartridege凝胶卡夹的安装及校正五、培训所需试剂、仪器及耗材六、DNA样品演示实验七、BioCalculator TM软件基本功能及数据分析八、常见问题解答一．QIAxcel全自动毛细管核酸分析系统介绍QIAGEN是全球核酸、蛋白质分离纯化试剂和新技术的权威供应商，总部设在德国。
其主打产品是QIAxcel，用于高通量DNA/RNA分离分析,这个产品广泛用于遗传分析，疾病检测以及食品鉴定等方面。
相比其它同类产品而言，其特点是高效，省时，安全，适用于标准化。
（一）产品介绍：技术参数：1．自动加样全自动化96孔加样；2．高通量12通道上样，96孔板连续检测；3．无需制胶更换式凝胶试剂盒；4．快速分析1－1.3小时/96个样品；5．高灵敏度PCR产物检测最低DNA浓度为0.1ng/ul；6．高分辨率500bp以下达2－5个bp；7．数据显示电泳图谱和峰图8．体积小便于搬运；9．污染小无敞开式EB污染。
10．重量23kg（二）BioCalculator TM软件1．操作简单，多种分离方法可供选择2．多种内嵌式方法模板适用各种用途3．数据分析界面友好：既可用于定性分析也可用于定量分析，一次完成多组数据分析4．数字化实验数据采集：●实验数据以电泳峰图和胶图两种形式显示●可适时电泳峰图和胶图●电子化实验数据方便储存和传递二．QIAxcel全自动毛细管核酸分析系统安装和维护（一）安装条件1．位置要求：稳定水平的操作平台放置设备，远离震荡设备。
2．温度要求：25℃3．湿度要求：40％－95％4．电源：100-230V~, 50-60 Hz, 360W (VA)5．电脑要求：●Processor处理器：Pentium IV 1.8 或更高●硬盘：40GB●内存：512 MB RAM●显示器：800x600●操作系统：Microsoft® Windows® XP6．氮气要求：纯度为99.999%的高纯氮气7．（二）维护用湿布清洁仪器的外表面。
基于宏基因组测序技术的非靶向筛查方法检测肉类食品中的动物源性成分
基于宏基因组测序技术的非靶向筛查方法 检测肉类食品中的动物源性成分丁清龙，杨丹婷，谢爱华，韦 云，陈秀芬，周 露*（广东省食品检验所，广东广州 510435）摘 要：目的：建立肉类食品中动物源性成分非靶向筛查方法。
方法：基于宏基因组测序技术建立肉类食品中动物源性成分非靶向筛查方法，将该方法用于模拟样品和实际样品检测，并用现有标准检测方法对实际样品检测结果进行确认。
结果：模拟样品检测结果与模拟情况一致；在实际样品中检出与样品名称不一致或样品标签未标识的动物源性成分，且非靶向筛查方法检测结果与现有标准方法检测结果一致。
结论：该方法可以快速锁定样品中未知的动物源性成分，检测结果准确可靠，可为政府打击肉类食品掺假提供更为有力的技术支撑。
关键词：宏基因组测序；动物源性成分；非靶向；掺假Determination of Animal-Derived Ingredients in Meat Products by Non-Targeted Screening Method Based on MetagenomicSequencing TechnologyDING Qinglong, YANG Danting, XIE Aihua, WEI Yun, CHEN Xiufen, ZHOU Lu*(Guangdong Institute of Food Inspection, Guangzhou 510435, China)Abstract: Objective: To establish the non-targeted screening method for animal-derived ingredients in meat products. Method: The non-targeted screening method for animal-derived ingredients in meat products was established based on metagenomic sequencing technology. The method was applied to the determination of simulated samples and actual samples, and the results of actual samples were confirmed by existing standard determination methods. Result: The determination results of simulated samples were consistent with design of simulated samples. The animal-derived ingredients inconsistent with sample name or label identification were detected in actual samples, and the results of non-targeted screening method and existing standard methods were consistent. Conclusion: This method could quickly test the unknown animal-derived ingredients of samples, and the determination results were accurate and reliable. It could provide more powerful technical support to combat meat adulteration for the government.Keywords: metagenomic sequencing technology; animal-derived ingredients; non-targeted; adulteration肉类食品是人们餐桌必备的主要食品之一，常见的高经济价值的肉类食品有牛肉、羊肉及其制品等。
QIAGEN质粒大提试剂盒
产品简介本试剂盒采用独特的硅胶模吸附技术，高效专一地结合质粒DNA。
同时采用特殊的溶液P4和过滤器CS1，可有效的出去内毒素、蛋白质杂志；整个提取过程仅需1h，方便快捷。
使用本试剂盒提取的质粒DNA可适用于各种常规操作，包括酶切、PCR、测序、连接、转化和细胞转染多种细胞等试验。
推荐每次菌液使用量：高拷贝质粒推荐使用量为100ml，得率一般在500-1500μg左右；低拷贝质粒推荐使用量为200ml，得率一般在200-600μg左右。
注意事项：请务必在使用本试剂盒之前阅读此注意事项。
1.溶液P1在使用前先加入RNase A （将试剂盒中提供的RNase A全部加入)，混匀，置于2-8℃保存。
2.第一次使用前应按照试剂瓶标签的说明先在漂洗液PW中加入无水乙醇。
3.使用前先检查平衡液BL，溶液P2和P4是否出现结晶或者沉淀，如有结晶或者沉淀现象，可在37℃水浴中加热几分钟，即可恢复澄清。
4.注意不要直接接触溶液P2和P4，使用后应立即盖紧盖子。
5.使用过滤器时请将推柄小心缓慢地从过滤管中抽出，避免滤膜因压力而松动。
6.提取的质粒量与细菌培养浓度，质粒拷贝数等因素有关。
如果所提质粒为低拷贝质粒或大于10kb的大质粒，应加大菌体使用量，同时按比例增加P1、P2、P4的用量；洗脱缓冲液推荐在65-70℃水浴中预热，可以适当延长吸附和洗脱时间，以提高提取效率。
7.实验前使用平衡液BL处理吸附柱，可以最大限度激活硅基质膜，提高得率。
8.用平衡液处理过的柱子最好立即使用，放置时间过长会影响使用效果。
质粒DNA 浓度及纯度检测得到的质粒DNA可用琼脂糖凝胶电泳和紫外分光光度计检测浓度与纯度。
OD260值为1相当于大约50μg/ml双链DNA。
纯化的质粒DNA OD260/OD280通常在1.8-2.0左右，可直接应用于细胞转染甚至动物体内实验等对DNA纯度要求很高的实验中。
操作步骤：1.柱平衡步骤：向吸附柱CP6中（吸附柱放入50ml收集管中）加入2.5ml的平衡液BL，8000rpm（~8228×g）离心2min，倒掉收集管中的废液，将吸附柱重新放回收集管中。
QIAGEN_OneStep_RT-PCR_Kit_Handbook
ContentsKit Contents4 Shipping and Storage Conditions4 Technical Assistance5 Product Warranty and Satisfaction Guarantee5 Product Use Limitations5 Safety Information6 Quality Control6 Product Specification7 Introduction8 QIAGEN OneStep RT-PCR Enzyme Mix8 QIAGEN OneStep RT-PCR Buffer9 Q-Solution9 Equipment and Reagents to be Supplied by user11 Protocols UsingQIAGEN OneStep RT-PCR Kit12 QIAGEN OneStep RT-PCR Kit and Q-Solution15 Troubleshooting Strategy19 Appendix A: Starting template24 Appendix B: Primer design, concentration, and storage25 Appendix C: Number of cycles 30 Appendix D: Sensitive RT-PCR assays 31 Appendix E: Touchdown PCR 31 Appendix F: Amplification of long RT-PCR products31 Appendix G: Multiplex RT-PCR 32 Appendix H: Co-amplification of an internal control 33 Appendix I: Purification of RT-PCR products 34 Appendix J: Control of contamination34 Appendix K: General remarks for handling RNA36 Ordering Information38QIAGEN OneStep RT-PCR Kit Handbook 10/201034QIAGEN OneStep RT-PCR Kit Handbook 10/2010QIAGEN OneStep RT-PCR Kit Handbook 10/201056QIAGEN OneStep RT-PCR Kit Handbook 10/2010Product SpecificationsEnzymes:The QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend for both reverse transcription and PCR. The unique combination of Omniscript and Sensiscript Reverse Transcriptases, with their high affinity for RNA templates, ensures highly efficient and sensitive transcription of RNA amounts from as little as 1 pg up to 2 µgOmniscript and Sensiscript Reverse Transcriptases are new, unique enzymes, and are different from the reverse transcriptases of Moloney murine leukemia virus (MMLV) or avian myeloblastosis virus (AMV). Omniscript and Sensiscript Reverse Transcriptases are recombinant heterodimeric enzymes expressed in E. coli.HotStarTaq DNA Polymerase is a chemically modified form of a recombinant 94-kDa DNA polymerase (deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), originally isolated from Thermus aquaticus, expressed in E. coli.Buffers and reagents:Storage buffer containing QIAGEN 20 mM Tris·Cl, 100 mM KCl, OneStep RT-PCR Enzyme Mix: 1 mM dithiothreitol (DTT), 0.1 mMEDTA, 0.5% (v/v) Nonidet®P-40,0.5% (v/v) Tween®20,50% glycerol (v/v), stabilizer;pH 9.0 (20°C)QIAGEN OneStep RT-PCR Buffer:5x concentrated. Contains Tris·Cl,KCl, (NH4)2SO4, 12.5 mM MgCl2,DTT; pH 8.7 (20°C)Q-Solution:5x concentrateddNTP Mix:10 mM each of dATP, dCTP, dGTP,and dTTP; PCR-gradeRNase-free water:Ultrapure quality, PCR-grade QIAGEN OneStep RT-PCR Kit Handbook 10/20107IntroductionThe QIAGEN OneStep RT-PCR Kit provides a convenient format for highly sensitive and specific RT-PCR using any RNA. The kit contains optimized components that allow both reverse transcription and PCR amplification to take place in what is commonly referred to as a “one-step” reaction.QIAGEN OneStep RT-PCR Enzyme MixThe QIAGEN OneStep RT-PCR Enzyme Mix contains a specially formulated enzyme blend specific for both reverse transcription and PCR amplification (see Table 1).I Omniscript and Sensiscript Reverse Transcriptases are included in the QIAGENOneStep RT-PCR Enzyme Mix and provide highly efficient and specific reverse transcription. Both reverse transcriptases exhibit a higher affinity for RNA, facilitating transcription through secondary structures that inhibit other reverse transcriptases. Omniscript Reverse Transcriptase is specially designed for reverse transcription of RNA amounts greater than 50 ng, and Sensiscript Reverse Transcriptase is optimized for use with very small amounts of RNA (<50 ng). This special enzyme combination in the QIAGEN OneStep RT-PCR Enzyme Mix provides highly efficient and sensitive reverse transcription of any RNA quantity from 1 pg to 2 µg.I HotStarTaq DNA Polymerase included in the QIAGEN OneStep RT-PCR EnzymeMix provides hot-start PCR for highly specific amplification. During reverse transcription, chemically modified HotStarTaq DNA Polymerase is completely inactive and does not interfere with the reverse-transcriptase reaction. After reverse transcription by Omniscript and Sensiscript Reverse Transcriptases, reactions are heated to 95°C for 15 min to activate HotStarTaq DNA Polymerase and to simultaneously inactivate the reverse transcriptases. This hot-start procedure using HotStarTaq DNA Polymerase eliminates extension from nonspecifically annealed primers and primer–dimers in the first cycle ensuring highly specific and reproducible PCR.Although all of the enzymes are present in the reaction mix, the use of HotStarTaq DNA Polymerase ensures the temporal separation of reverse transcription and PCR allowing both processes to be performed sequentially in a single tube. Only one reaction mix needs to be set up: no additional reagents are added after the reaction starts.8QIAGEN OneStep RT-PCR Kit Handbook 10/2010QIAGEN OneStep RT-PCR BufferQIAGEN OneStep RT-PCR Buffer is designed to enable both efficient reverse transcription and specific amplification.I The unique buffer composition allows reverse transcription to be performed at hightemperatures (50°C). This high reaction temperature improves the efficiency of the reverse-transcriptase reaction by disrupting secondary structures and is particularly important for one-step RT-PCR performed with limiting template RNA amounts.I It has been reported that one-step RT-PCR may exhibit reduced PCR efficiencycompared to two-step RT-PCR. The combination of QIAGEN enzymes and the unique formulation of the QIAGEN OneStep RT-PCR Buffer ensures high PCR efficiency in one-step RT-PCR.I The buffer contains the same balanced combination of KCl and (NH4)2SO4includedin QIAGEN PCR Buffer. This formulation enables specific primer annealing over a wider range of annealing temperatures and Mg2+concentrations than conventional PCR buffers.†The need for optimization of RT-PCR by varying the annealing temperature or the Mg2+concentration is therefore minimized.Q-SolutionThe QIAGEN OneStep RT-PCR Kit is provided with Q-Solution, an innovative additive that facilitates amplification of difficult templates by modifying the melting behavior of nucleic acids.I Q-Solution often enables or improves suboptimal RT-PCR caused by RNA and DNAtemplates that have a high degree of secondary structure or that are GC-rich.I Unlike other commonly used additives such as DMSO, Q-Solution is used at just oneworking concentration. F or further information, please read the Protocol Using QIAGEN OneStep RT-PCR Kit and Q-Solution Protocol, page 15.QIAGEN OneStep RT-PCR Kit Handbook 10/20109QIAGEN OneStep RT-PCR ProcedureThe QIAGEN OneStep RT-PCR Kit allows fast and easy RT-PCR setup. Whatever your application — virus detection, molecular diagnostics research, or gene expression — just mix all components together in one tube and start your thermal cycler program (see flowchart).QIAGEN OneStep RT-PCR Procedure10QIAGEN OneStep RT-PCR Kit Handbook 10/2010Equipment and Reagents to be Supplied by userPrimers:The QIAGEN OneStep RT-PCR Kit is designed to be used with gene-specific primers. The use of random oligomers or oligo-dT primersis not recommended.RNase inhibitor*:RNase inhibitor is a 50 kDa protein that strongly inhibits RNases A, B, (optional)and C, as well as human placental RNases. It helps to minimize the risk of RNA degradation during experimental setup.Protocol Using QIAGEN OneStep RT-PCR KitThis protocol serves as a guideline for one-step RT-PCR. Reverse transcription and PCR are carried out sequentially in the same tube. All components required for both reactions are added during setup, and there is no need to add additional components once the reaction has been started. The protocol has been optimized for 1 pg – 2 µg of total RNA. Optimal reaction conditions, such as incubation times and temperatures during PCR amplification, will vary and need to be determined individually.Important notes before startingI HotStarTaq DNA Polymerase, contained in the QIAGEN OneStep RT-PCR Enzyme Mix,requires initial activation by incubation at 95°C for 15 min before amplification can take place (see step 6 of this protocol). This incubation also inactivates the reverse transcriptases. Do not heat activate the HotStarTaq DNA Polymerase until the reverse-transcriptase reaction is finished.I The QIAGEN OneStep RT-PCR Kit is designed to be used with gene-specific primersat a final concentration of 0.6 µM. The use of random oligomers or oligo-dT primers is not recommended since it will result in the amplification of nonspecific products.I Set up all reactions on ice.I Make sure the thermal cycler is preheated to 50°C before placing samples in it.I The 5x QIAGEN OneStep RT-PCR Buffer provides a final concentration of 2.5 mMMgCl2in the reaction mix, which will produce satisfactory results in most cases.I An RNase-free environment should be maintained during RNA isolation and reactionsetup.I Set up the reaction mixtures in an area separate from that used for RNA preparationor PCR product analysis.I Use disposable tips containing hydrophobic filters to minimize cross-contamination.Procedure1.Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-PCRBuffer, and RNase-free water, and place them on ice.It is important to mix the solutions completely before use to avoid localized differences in salt concentration.2.Prepare a master mix according to Table 2.The master mix typically contains all the components required for RT-PCR except the template RNA. Prepare a volume of master mix 10% greater than that required for the total number of reactions to be performed. A negative control (without template RNA) should be included in every experiment (see Appendix J, page 34).Table 2. Reaction components for one-step RT-PCRComponent Volume/reaction Final concentration Master mixRNase-free water (provided)Variable–5x QIAGEN OneStep RT-PCR Buffer*10.0 µl1xdNTP Mix (containing 10 mM 2.0 µl400 µM of each dNTPof each dNTP)Primer A Variable0.6 µM†Primer B Variable0.6 µM†QIAGEN OneStep RT-PCR Enzyme Mix 2.0 µl–RNase inhibitor (optional)‡Variable5–10 units/reaction Template RNATemplate RNA, added at step 4Variable 1 pg – 2 µg/reaction Total volume50.0 µl–*Contains 12.5 mM MgCl2† A final primer concentration of 0.6 µM is optimal for most primer–template systems. However, in some cases using other primer concentrations (i.e., 0.5–1.0 µM) may improve amplification performance.‡The use of RNase inhibitor is optional; because the buffer composition has an inhibitory effect on RNAses.3.Mix the master mix thoroughly, and dispense appropriate volumes into PCR tubes.Mix gently, for example, by pipetting the master mix up and down a few times. 4. Add template RNA (Յ2 µg/reaction) to the individual PCR tubes.The QIAGEN OneStep RT-PCR Kit can be used with total RNA, messenger RNA, or viral RNA.5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directlyto step 6. Otherwise, overlay with approximately 50 µl mineral oil.6. Program the thermal cycler according to the program outlined in Table 2, page 13.Table 2 describes a typical thermal cycler program. The program includes steps for both reverse transcription and PCR. The PCR amplification segment must start with an initial heating step at 95°C for 15 min to activate HotStarTaq DNA Polymerase. For maximum yield and specificity, temperatures and cycling times can be further optimized for each new target and primer pair. However, the protocol gives satisfactory results in most cases.7. Start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cyclerhas reached 50°C. Then place the PCR tubes in the thermal cycler.Note:After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer-term storage.Table 3. Thermal cycler conditionsAdditional commentsReverse transcription:30 min50°C A reverse-transcription reactiontemperature of 50°C is recom-mended. However, if satisfactoryresults are not obtained using50°C, the reaction temperaturemay be increased up to 60°C. Initial PCR activation step:15 min95°C HotStarTaq DNA Polymerase isactivated by this heating step.Omniscript and Sensiscript ReverseTranscriptases are inactivated andthe cDNA template is denatured.3-step cyclingDenaturation:0.5–1 min94°CAnnealing:0.5–1 min50–68°C Approximately 5°C below T ofprimers.Extension: 1 min72°C F or RT-PCR products of 1–2 kb,increase the extension time by30–60 s. For RT-PCR products over2 kb, see appendix, page 31. Number of cycles:25–40The cycle number is dependenton the amount of templateRNA and the abundance of thetarget transcript. See Appendix C,page 30.Final extension:10 min72°CProtocol Using QIAGEN OneStep RT-PCR Kit and Q-SolutionThis protocol is designed for using Q-Solution in one-step RT-PCR. Q-Solution changes the melting behavior of nucleic acids and can be used for RT-PCR systems that do not work well under standard conditions. When using Q-Solution the first time with a particular primer–template system, always perform parallel reactions with and without Q-Solution. This recommendation should also be followed if another RT-PCR additive (such as DMSO) was previously used for a particular primer–template system. When using Q-Solution, the following effects may be observed depending on the indi-vidual RT-PCR assay:Case A:Q-Solution enables RT-PCR that previously failed.Case B:Q-Solution increases RT-PCR specificity in certain primer–template systems.Case C:Q-Solution has no effect on RT-PCR performance.Case D:Q-Solution causes reduced efficiency or failure of a previously successfulamplification reaction. In this case, addition of Q-Solution disturbs the previously optimal primer–template annealing. Therefore, when using Q-Solution for the first time for a particular primer–template system, always perform reactions in parallel with and without Q-Solution.439 bp874 bp1.4 kb690 bpM ––++M ––++M ––++M ––++–:without Q-Solution +:with Q-Solution M:markersDImportant notes before startingI HotStarTaq DNA Polymerase, contained in the QIAGEN OneStep RT-PCR Enzyme Mix,requires initial activation by incubation at 95°C for 15 min before amplification can take place (see step 6 of this protocol). This incubation also inactivates the reverse transcriptases. Do not heat activate the HotStarTaq DNA Polymerase until the reverse-transcriptase reaction is finished.I Q-Solution modifies the melting behavior of nucleic acids and can be used forprimer–template systems that do not perform well using standard conditions. When using Q-Solution the first time for a particular primer–template system, always perform parallel reactions with and without Q-Solution.I The QIAGEN OneStep RT-PCR Kit is designed for use with gene-specific primers ata final concentration of 0.6 µM. The use of random oligomers or oligo-dT primersis not recommended since it will result in the amplification of nonspecific products.I Set up all reactions on ice.I Make sure the thermal cycler is preheated to 50°C before placing samples in it.I The 5x QIAGEN OneStep RT-PCR Buffer provides a final concentration of 2.5 mMMgCl2in the reaction mix, which will produce satisfactory results in most cases.I An RNase-free environment should be maintained during RNA isolation and reactionsetup.I Set up reaction mixtures in an area separate from that used for RNA preparation orPCR product analysis.I Use disposable tips containing hydrophobic filters to minimize cross-contamination.Procedure1.Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-PCRBuffer, Q-Solution, and RNase-free water, and place them on ice.It is important to mix the solutions completely before use to avoid localized differences in salt concentration.2.Prepare a master mix according to Table 4.The master mix typically contains all the components required for RT-PCR except the template RNA. Prepare a volume of master mix 10% greater than that required for the total number of reactions to be performed. A negative control (without template RNA) should be included in every experiment (see Appendix J, page 34).When using Q-Solution for the first time for a particular primer–template system, always perform parallel reactions with and without Q-Solution.3.Mix the master mix thoroughly, and dispense appropriate volumes into PCR tubes.Mix gently, for example, by pipetting the master mix up and down a few times.Table 4. Reaction components for one-step RT-PCR using Q-SolutionComponent Volume/reaction Final concentration Master mixRNase-free water (provided)Variable–5x QIAGEN OneStep RT-PCR Buffer*10.0 µl1xdNTP Mix (containing 10 mM 2.0 µl400 µM of each dNTPof each dNTP)5x Q-Solution 10.0 µl1xPrimer A Variable0.6 µM†Primer B Variable0.6 µM†QIAGEN OneStep RT-PCR Enzyme Mix 2.0 µl–RNase inhibitor (optional)‡Variable5–10 units/reaction Template RNATemplate RNA, added at step 4Variable 1 pg – 2 µg/reaction Total volume50.0 µl–*Contains 12.5 mM MgCl2† A final primer concentration of 0.6 µM is optimal for most primer–template systems. However, in some cases using other primer concentrations (i.e., 0.5–1.0 µM) may improve amplification performance.‡The use of RNase inhibitor is optional because the buffer composition has an inhibitory effect on RNAses. 4.Add template RNA (Յ2 µg/reaction) to the individual PCR tubes.The QIAGEN OneStep RT-PCR Kit can be used with total RNA, messenger RNA, or viral RNA.5.When using a thermal cycler with a heated lid, do not use mineral oil. Proceed directlyto step 6. Otherwise, overlay with approximately 50 µl mineral oil.6.Program the thermal cycler according to the program outlined in Table 5, page 18.Table 4 describes a typical thermal cycler program. The program includes steps for both reverse transcription and PCR. The PCR amplification segment must start with an initial heating step at 95°C for 15 min to activate HotStarTaq DNA Polymerase. For maximum yield and specificity, temperatures and cycling times should be optimized for each new target and primer pair.7.Start the RT-PCR program while PCR tubes are still on ice. Wait until the thermal cyclerhas reached 50°C. Then place the PCR tubes in the thermal cycler.Note:After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer-term storage.Table 5. Thermal cycler conditionsAdditional commentsReverse transcription:30 min50°C A reverse-transcription reactiontemperature of 50°C is recom-mended. However, if satisfactoryresults are not obtained using50°C, the reaction temperaturemay be increased up to 60°C. Initial PCR activation step:15 min95°C HotStarTaq DNA Polymerase isactivated by this heating step.Omniscript and Sensiscript ReverseTranscriptases are inactivated andthe cDNA template is denatured.3-step cyclingDenaturation:0.5–1 min94°CAnnealing:0.5–1 min50–68°C Approximately 5°C below T m ofprimers.Extension: 1 min72°C F or RT-PCR products of 1–2 kb,increase the extension time by30–60 s. For RT-PCR products over2 kb, see Appendix B, page 29. Number of cycles:25–40The cycle number is dependenton the amount of templateRNA and the abundance of thetarget transcript. See Appendix C,page 30.Final extension:10 min72°CTroubleshooting GuideThis troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: /F AQ/F AQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies (for contact information, see back cover or visit ).Comments and suggestionsLittle or no producta) Pipetting error or missing reagent Check the concentrations and storageconditions of reagents, including primers anddNTP Mix. Repeat the RT-PCR.b)HotStarTaq DNA Polymerase Ensure that the cycling program included thenot activated HotStarTaq DNA Polymerase activation step(15 min at 95°C) as described in step 6 of theprotocols (pages 13 and 17).c)HotStarTaq DNA Polymerase Check the cycling program. Ensure that theactivated too early reverse-transcription reaction is complete(30 min at 50°C) before activating theHotStarTaq DNA Polymerase (15 min at 95°C).d) Reverse-transcription reaction A reverse-transcription reaction temperaturetemperature incorrect of 50°C is recommended. However, ifdesired results are not obtained using 50°C,reaction temperatures of 45–60°C may beused.e) Primer concentration not optimal A primer concentration of 0.6 µM is stronglyor primers degraded recommended. However, if the desired resultsare not obtained using this concentration,repeat the RT-PCR with different primerconcentrations from 0.5–1.0 µM in 0.1 µMincrements. In particular, when performinghighly sensitive RT-PCR, check for possibledegradation of the primers on a denaturingpolyacrylamide gel*.*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs),available from the product supplier.*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs), available from the product supplier.*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs), available from the product supplier.Appendix A: Starting templateThe efficiencies of reverse transcription and PCR are highly dependent on the quality and quantity of the starting RNA template.It is important to have intact RNA as starting template. Even trace amounts of contaminating RNases in the RNA sample can cause RNA cleavage, resulting in shortened cDNA products. Chemical impurities, such as protein, poly-anions (e.g., heparin), salts, EDTA, ethanol, phenol, and other solvents, can affect the activity and processivity of the reverse transcriptases and the Taq DNA polymerase. To ensure reproducible and efficient RT-PCR, it is important to determine the quality and quantity of the starting RNA.For best results, we recommend starting with RNA purified using silica-gel–membrane technology. For example, RNeasy®Kits, QIAamp®Viral RNA Kits, and the QIAamp RNA Blood Mini Kit can be used to isolate RNA from a variety of starting materials and provide high-quality RNA ideal for use in reverse-transcription and RT-PCR applications. Alternatively RNA can be isolated from whole blood using the PAXgene Blood RNA Kit. Storage of RNAPurified RNA may be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable for at least 1 year.Determining concentration and purity of RNAI The concentration of RNA should be determined by measuring the absorbance at260 nm (A260) in a spectrophotometer. Note that absorbance measurements cannot discriminate between DNA and RNA.I To determine RNA concentration, we recommend dilution of the sample in watersince the relationship between absorbance and concentration (A260reading of1 = 40 µg/ml RNA) is based on an extinction coefficient calculated for RNA inwater. To ensure significance, readings should be between 0.1 and 1.0.I The ratio between the absorbance values at 260 and 280 nm gives an estimateof RNA purity. To determine RNA purity, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A260/A280ratio of 1.9–2.1* in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the spectrophotometer using the same solution.*Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.*When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs), available from the product supplier.Exon Exon IntronGenomic DNAmRNA No productRT-PCRExon Exon IntronGenomic DNAmRNA Exon RT-PCRRT-PCR Large product Small productFigure 1. Primer design for RT-PCR.Primer design to A eliminate or B detect amplification from contaminating genomic DNAPrimer spans an intron/exon boundaryPrimers flank an intronAppendix D: Sensitive RT-PCR assaysRT-PCR can be performed to detect even a single RNA molecule. However, amplification of such a low number of target sequences is often limited by the generation of nonspecific RT-PCR products and primer–dimers. The combination of HotStarTaq DNA Polymerase and QIAGEN OneStep RT-PCR Buffer increases specificity in the first cycle and throughout PCR. Thus HotStarTaq DNA Polymerase is well suited to such highly sensitive RT-PCR assays.Nested PCRIf PCR sensitivity is too low, a nested PCR method can increase RT-PCR product yield. Nested RT-PCR involves reverse transcription followed by two rounds of amplification reactions. The first-round PCR is performed according to the QIAGEN OneStep RT-PCR Protocol (page 10 or 13). Subsequently, an aliquot of the first-round PCR product, for example 1 µl of a 1/103– 1/104dilution, is subjected to a second round of PCR. The second-round PCR is performed using two new primers that hybridize to sequences nternal to the first-round primer target sequences. In this way, only specific first-round RT-PCR products (and not nonspecific products) will be amplified in the second round. Alternatively, it is possible to use one internal and one first-round primer in the second PCR; this method is referred to as semi-nested PCR.Appendix E: Touchdown PCRTouchdown PCR uses a cycling program with varying annealing temperatures. It is a useful method to increase the specificity of RT-PCR. The annealing temperature in the initial PCR cycle should be 5–10°C above the T m of the primers. In subsequent cycles, the annealing temperature is decreased in steps of 1–2°C per cycle until a temperature is reached that is equal to, or 2–5°C below, the T m of the primers. Touchdown PCR enhances the specificity of the initial primer–template duplex formation and hence the specificity of the final RT-PCR product.To program your thermal cycler for touchdown PCR, you should refer to the manufacturer’s instructions.Appendix F: Amplification of long RT-PCR productsThe QIAGEN OneStep RT-PCR Protocol is optimized for amplification of products of up to 2 kb. When amplifying RT-PCR products larger than 2 kb, it is recommended to use the modified cycling conditions described in Table 12, page 32.For amplification of very long RT-PCR products of up to 12.5 kb, we recommend the QIAGEN LongRange 2 Step RT-PCR Kit.。
QIAGEN DNeasy Blood & Tissue 试剂盒说明书(翻译版)
使用前注意事项：⏹所有离心操作在室温（15℃-25℃）下进行⏹沉渣用Buffer AL和Buffer ATL重溶⏹在浓缩的Buffer AW1和Buffer AW2中加入乙醇⏹冰冻组织和细胞球在室温下平衡⏹在56℃孵育箱中预处理⏹固定组织、昆虫、细菌或其他材料的预处理请参见使用手册1a. 组织：将组织切碎成小块（脾脏≤10mg，其他组织≤25mg），装入1.5ml离心管中，鼠尾则取1cm（大鼠）或2cm（小鼠）。
加入180μl Buffer ATL。
加入20μl 蛋白激酶K，振荡混匀，56℃孵育至组织完全裂解，孵育过程中间断振荡。
进入步骤2前振荡15秒。
1b. 无核血液：将20μl蛋白激酶K移至1.5ml或2ml离心管，加入50-100μl 抗凝处理过的血液。
用PBS调整体积至220μl，进入步骤2。
1c. 有核血液：将20μl蛋白激酶K移至1.5ml或2ml离心管，加入5-10μl抗凝处理过的血液。
用PBS调整体积至220μl，进入步骤2。
1d. 培养细胞：最多可用5×106个细胞，300×g（190rpm）离心5min。
用200μl重悬，加入20μl蛋白激酶K进入步骤22.加入200μl Buffer AL，振荡混匀，血标本需在56℃下孵育10min。
3.加入200μl 乙醇，振荡混匀。
4.将上述混合物用移液枪移至2ml离心管中的DNeasy Mini 滤柱中，6000×g（8000rpm）离心1min，弃去滤出液及离心管。
5.将滤柱放入新的2ml收集管中，加入500μl Buffer AW1，≥6000×g离心1min，弃去滤出液及收集管。
6.将滤柱放入新的2ml收集管中，加入500μl Buffer AW2，20000×g（14000rpm）离心3min，弃去滤出液及收集管。
7.将滤柱移至新的1.5ml或2ml离心管。
8.将200μl Buffer AE加至滤柱膜中央以洗提DNA，室温（15℃-25℃）下孵育1分钟。