QIAGEN-DNeasy mericon food handbook

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-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。

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。

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。

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.。

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。

襄阳大头菜腌制液生物膜真菌多样性研究赵慧君;沈馨;董蕴;吴竞;凌霞;胡事成;郭壮【摘要】采集了3个襄阳大头菜腌制液生物膜样品,使用Miseq高通量测序技术对其真菌微生物群落结构进行了解析.在门水平上,Ascomycota平均含量高达99.99％,相对含量大于1％的真菌属分别为Zygosaccharomyces,Pichia和Candida,其平均相对含量分别为56.10％,40.96％和2.32％.在分类操作单元(operational taxonomic units,OTU)水平上,发现24个核心OTUs,其中OTU62(Zygosaccharomyces)和OTU37 (Phialosimplex)的累计平均相对含量为83.60％.由此可见,襄阳大头菜生物膜中的真菌微生物主要由隶属于Ascomycota的Zygosaccharom yces和Pichia构成,生物膜共有大量的核心真菌菌群.%In the current study,3 biomembrane samples of Xiangyang mustard root brineare collected,and the fungal microbiota are studied by Miseq high throughput sequencing technologies.Ascomycota is the most dominant fungal phylum with the relative abundance of 99.99％.At the phylum level,Zygosaccharomyces,Pichia and Candida are constituting more than 1％of total sequences,with the relative abundance of 56.10％,40.96％ and 2.32％respectively.At the OTU level,24 OTUs are shared by all samples,and the cumulative average relative content of OTU62(Zygosaccharomyces)and OTU37(Phialosimplex)is 83.60％.The results indicate that the fungal microbiota of biomembrane in Xiangyang mustard root brine are main consisted by Zygosaccharomyces and Pichia belong to Ascomycota,and biomembrane samples have a large number of core fungal flora.【期刊名称】《中国调味品》【年(卷),期】2017(042)012【总页数】5页(P61-65)【关键词】襄阳大头菜;生物膜;真菌;多样性;Miseq【作者】赵慧君;沈馨;董蕴;吴竞;凌霞;胡事成;郭壮【作者单位】湖北文理学院化学工程与食品科学学院鄂西北传统发酵食品研究所,湖北襄阳441053;湖北文理学院化学工程与食品科学学院鄂西北传统发酵食品研究所,湖北襄阳441053;湖北文理学院化学工程与食品科学学院鄂西北传统发酵食品研究所,湖北襄阳441053;襄阳市食品药品检验所,湖北襄阳441021;襄阳市食品药品检验所,湖北襄阳441021;襄阳孔明菜有限公司,湖北襄阳441000;湖北文理学院化学工程与食品科学学院鄂西北传统发酵食品研究所,湖北襄阳441053【正文语种】中文【中图分类】TS255.3作为我国四大名腌菜之一的襄阳大头菜是以芥菜为原料，采用独特的“三腌、五卤、六晒”工艺历时18个月腌制而成，该制作工艺至今已有近2000年的历史。

QIAGEN EpiTect® Bisulfite•重亚硫酸盐处理DNA时，需要经高盐浓度、高温和低PH条件，会导致DNA断裂和片段化，并在随后的纯化过程中丢失，所以需要大量的input DNA•重亚硫酸盐转化未甲基化的胞嘧啶，转化率超过99%•Bisulfite Mix 足够做8个反应，用时要将Bisulfite Mix溶于800μl RNase-free water中，溶解的Bisulfite Mix 在-20℃可以保存4周•DNA Protect Buffer 能保护重亚硫酸盐处理的DNA在高温、低ph条件下被片段化•Carrier RNA 能提高小量DNA（小于500pg，体积大于40μl）绑定在回收柱滤膜上的效率，帮助核酸沉淀；总量大于100ng的基因组DNA没有必要加Carrier RNA•EpiTect Bisulfite Kit 在-20℃可以保存3年；可以适用于大范围的DNA量，input DNA范围为1ng~2μg；模板DNA片段范围可以在500bp~30kb之间DNA量在1ng~2μg之间，体积20μl以上开始前的准备重点：•Bisulfite Mix 足够做8个反应，如果样本少于8个，可以把溶解的Bisulfite Mix 保存于-20℃，4周内并不会影响其性能•DNA Protect Buffer在加入DNA–Bisulfite Mix后会由绿变蓝（step 2），表明溶液混合很充分且PH值正确•所有的离心分离步骤均在室温（15–25°C）下完成开始前准备的东西：•加入30ml乙醇（96%~100%）到Buffer BW中，室温（15–25°C）保存，使用之前摇匀•加入27ml乙醇（96%~100%）到Buffer BD中，2~8℃保存，使用之前摇匀，使用之后要立即盖上盖子。

储存一段时间后底部可能会出现白色沉淀，沉淀不影响Buffer BD的性能，但是还是要避免将沉淀吸到回收柱中。

DNeasy® 96 Plant Handbook:For high-throughput DNA isolation from plant tissue (24-27)DNeasy® 96 Plant Protocol for Isolation of DNA from Fresh Plant Leaves Using the Mixer Mill MM 300（DNeasy 96植物技术：使用搅拌磨MM 300从新鲜植物叶片中分离DNA）一、Important notes before starting（使用前的重要注释）1、Take time to familiarize yourself with the Mixer Mill MM 300 and the Centrifuge4-15C/ Centrifuge 4K15C before starting this protocol（开始使用本实验方法之前花时间熟悉搅拌磨MM 300和离心机4-15C/ 离心机4K15C）。

2、This protocol describes processing of 192 samples (2×96). If you wish to process 96 or fewer samples, provide a balance for the mixer mill by assembling a second plate sandwich using a rack of collection microtubes without samples or buffers, but containing tungsten carbide beads, and fixing this second sandwich into the empty clamp （本方法用来对2×96=192个样本进行提取DNA，如果希望处理96个或更少样本，要对搅拌磨MM 300进行平衡处理，方法是用一架没有样本和缓冲液但装有碳化钨珠子的收集微型管插入第二个孔板，并用空夹子加入固定）。

Vol.33,No.6Dec. 2020第33卷第6期2020年12月水产学杂志CHINESE JOURNAL OF FISHERIES文章编号:1005-3832( 2020 )06-0028-06用DNA 条形码鉴定常见鱼翅的种类与真伪熊娟，黄启红，陈敏儿，方军(广东省科学院，广东省测试分析研究所(中国广州分析测试中心)，广东省化学危害应急检测技术重点实验室，广东省保健食品功效成分检测与风险物质快速筛查工程技术研究中心，广东广州510070)摘要:用形态鉴定了物种的市面上常见的6种正品鱼翅作标准样品，以明胶为主要原料制作的2种假鱼翅作阴 性对照，用DNA 条形码技术研究了广东省市场流通的35种鱼翅的遗传多样性，探讨从基因水平鉴别鱼翅制品真伪的方法，提高鱼翅产品标识准确性。

采用不同基因组DNA 提取方法比对，选择适合鱼翅DNA 的提取旗 筛选出C01序列DNA 条形码通用引物,对鱼翅DNA 进行PCR 扩增并测序,应用MEGA 6.0软件对64条鱼翅COZ 序列进行多序列比对，基于邻接法(N-J )构建进化树，分析遗传距离。

同时应用DNAman 构建相应COZ 序列的聚类同源树。

结果表明,本研究所建立的条形码技术能鉴别13种鱼翅物种。

分子系统发育树显示侗_个物种的不同个体基本都能形成单系，节点支持率大都为99% ~ 100%。

聚类分析显示侗一物种都位于同一个分支，不同个体的鱼类的CO/序列相似度均大于95%。

该方法可为广东省流通的鱼翅制品的鉴定提供分子生物 学基础。

关键词:鱼翅;DNA 条形码技术;鉴定中图分类号:S917文献标识码:AIdentification of Species and Authenticity of Shark Fins by DNA Barcoding TechnologyXIONG Juan, HUANG Qihong, CHEN Miner, FANG Jun(Guangdong Provincial Engineering Research Center for Efficacy Component Testing and Risk Substance Rapid Screening of H ealthFood, Guangdong Provincial Key Laboratory of E mergency Test for Dangerous Chemicals, Guangdong Institute of A nalysis ( ChinaNational Analytical Center Guangzhou), Guangdong Academy of Sciences, Guangzhou 510070, China)Abstract: Six species of authentic shark fins in the market were identified by morphology as standard samples, and two kinds of fake shark fins made with gelatin as the main raw material were used as negative controls. The genetic diversity of 35 types of shark fins in the market in Guangdong Province was investigated using DNA barcode technology to probe for a method of identifying the authentic ­ity of shark fin products from the genetic level with higher accuracy of identification of shark fins products in Guangdong Province. Different genomic DNA extraction methods were compared and the most suitable DNA extraction methods for shark fins were chosen.The universal primers of DNA barcode of COI sequence were screened out, and the DNA of shark fins was amplified and sequenced by PCR. The multiple sequence alignment of 64 shark fin COI sequences was carried out by MEGA 6.0 software. The phylogenetictree was constructed based on the Neighbor-Joining method (N-J ), and the genetic distance was analyzed, and DNAman was used to construct cluster homology tree of COI sequences, with 13 species of shark fin being identified by the DNA barcode technology.Molecular phylogenetic tree showed that diSerent individuals of the same species had basically monophy form, with support rate of 99%〜100% for most of the nodes. Cluster analysis revealed that the same species was located in the same branch, and that the COI se ­quence similarity of fishes with different individuals was all greater than 95%. In conclusion, this method provides molecular biological basis for identification of shark fins products in Guangdong Province.Key words: shark fin; DNA barcode technology; identification广东省素被誉为养生大省，而鱼翅作为广东著 度水涨船高。

QIAGEN试剂盒提取RNA说明书中⽂翻译1:细菌的溶菌酶破碎2:细菌的溶菌酶破碎和机械破碎3:细菌的机械破碎4:细菌的溶菌酶裂解和蛋⽩酶K的消化5:细菌的溶菌酶裂解、蛋⽩酶K的消化及机械破碎6:固体培养基细菌的裂解7:使⽤RNeasy Mini Kit从细菌溶解物中提纯总RNA。

8:使⽤RNeasy Midi Kit从细菌溶解物中提纯总RNA。

章节说明该⼿册含有两种不同类型⽅案。

我们提供了多种⽅案，其中⽅案1-6是针对制备细菌溶解物，⽅案7-8是针对如何从细菌溶解物中提取全RNA。

你需要在⽅案1-6中选择⼀种操作，⽽后在⽅案7-8中任选其⼀。

制备细菌溶解物的各个⽅案都介绍了破碎细菌时如何固定RNA。

如何选择不同的实验⽅案由细菌细胞壁的稳定性所决定。

细菌细胞壁的稳定性受多个因素影响，包括细菌种类、⽣长阶段和培养基的成分。

细菌必须完全破碎以确保RNA的有效提取。

制备细菌溶解物的各个⽅案都包含了不⽌⼀步。

■酶消化：细菌细胞壁可以被溶菌酶进⾏消化（例如溶菌酶、溶葡球菌酶）。

我们认为溶菌酶的作⽤对于所有的⾰兰⽒阳性、阴性细菌均是有效的。

■蛋⽩酶K消化：在复杂培养基上⽣长的细菌⼤部分蛋⽩质可以被蛋⽩酶K消化以提⾼RNA 的纯化率。

我们认为蛋⽩酶K对于可在复合培养基中⽣长的细菌均是有效的。

蛋⽩酶K常常⽤于提⾼⾰兰⽒阳性细菌RNA。

另外，若从⼤量原材料中提取RNA，蛋⽩酶K可以有效提⾼RNA产率。

■机械破碎：细菌细胞壁的机械破碎可以使⽤组织研磨机和玻璃微珠。

机械破碎法对于绝⼤多数的细菌来说都是适⽤的。

机械破碎法与酶消化法相结合可以极⼤程度的提⾼RNA产率。

尽管本⼿册中的机械破碎⽅案均为使⽤组织研磨机，但采⽤其他的破碎⽅法是完全可⾏的。

考虑到细菌种类的多样性以及培养条件的多样性，细菌溶解物的制备⽅案（⽅案1-6）必须谨慎选择。

在第10页中介绍了制备细菌溶解物的不同⽅案，第11页（表格⼀）则提供了这些⽅案概况以便于参考选择。

miScript PCR 系统操作手册试剂盒成分：miScript Reverse Transcription KitmiScript Primer AssaysmiScript SYBR® Green PCR Kit10x miScript Primer Assay 和10x miScript Precursor Assay是干粉状的，需要稀释。

首先短暂离心小管，然后加入550μl TE，pH 8.0，最后在漩涡震荡仪上4-6次。

为了保持引物的活性，在冷冻条件下将稀释的引物分装成小管。

实验步骤：反转录反应1、在冰上融化RNA，室温下(15–25ºC)融化5x miScript RT Buffer 和RNasefree water 。

先轻弹每个小管使其充分混匀，然后短暂离心以收集管壁和管盖上的液体，最后放置在冰上。

2、在冰上配制reverse-transcription master mix。

配好后混合并放置在冰上。

如果miScript Reverse Transcriptase Mix是从-20°C 冰箱拿出来的话，那么在使用完毕后立刻放入冰箱。

如果要配制多次反应，先在一个大管中准备Mix，并且多配制10%。

3、将miScript Reverse Transcriptase Mix分装到每个小管中，然后加入RNA。

4、在37°C下孵育60分钟5、在95°C下孵育5分钟以灭活miScript Reverse TranscriptaseMix6、如果马上进行PCR，讲产物放置在冰上；如果不立刻做PCR，那么将反转录产物放置在-20°C实验步骤：Real-Time PCR检测miRNA或Noncoding RNA1、融化2x QuantiTect SYBR Green PCR Master Mix，10x miScriptUniversal Primer, 10x miScript Primer Assay, template cDNA, 和RNase-free water。

纯化高转染级别的质粒DNA 【工作台流程】BENCH PROTOCOL准备工作：1.将RNase A 溶液加入P1缓冲液(Buffer P1)2.加40ml96－100％乙醇(ethanol)于试剂盒的去内毒素水中(endotoxin-fre water)3.可选：加 LyseBlue 试剂于 Buffer P14.检查 Buffer P2是否有SDS沉淀precipitation 有沉淀可在37摄氏度下暖化溶解5.Buffer P3 置于4摄氏度中预冷pre-chill6.细菌扩增培养补充：1.准备4-6个50ml新的或灭菌的离心管用于冷冻离心机下离心过夜菌液，2个用于接下来的菌块重悬和与buffer混合2.1个无内毒素的30ml聚丙烯（不推荐聚碳酸酯，因为不耐乙醇）管子（最好用圆底的离心管以利于高速离心）用于收集洗提的质粒DNA3.5个1.5ml的EP管用于收集抽提过程各步骤的产物用于实验后分析和质量控制4.准备1个冰浴盒用于步骤65.准备室温下的异丙醇10.5ml6.4摄氏度冰冻离心机和分光光度仪,琼脂糖凝胶电泳步骤：peocedureA.细菌培养、收获和裂解bacterial culture,harvest & lysis1.100ml高拷贝high-copy或250ml低拷贝low-copy过夜overnight LB培养菌液于冰冻离心机4摄氏度；6000×g；15min从新鲜的抗生素平板上挑取一个单菌落，2-5ml抗生素LB液体初始培养基30摄氏度孵化约8小时（振荡300rpm，注意孵化容器容积至少4倍于培养液）抗生素LB液体培养基稀释初始培养液到1:500-1000。

(高拷贝质粒100-200ul初始培养液加于100ml培养基；低拷贝质粒250-500ul初始培养液加于250ml培养基)37摄氏度300rpm振荡12-16小时。

(孵化容器容积至少4倍于培养液，培养至细菌密度约3-4×109/ml，即离心后菌块湿重约3g/L培养基)培养基配方：10g蛋白胨+5g酵母+10g氯化钠溶于800ml蒸馏水，用1 N NaOH 调pH值至7.0，用蒸馏水调整至1L。

DNeasy®Plant HandbookDNeasy Plant Mini KitDNeasy Plant Maxi KitFor purification of total cellular DNA from plant cells and tissues,or fungiDNeasy96Plant KitFor high-throughput purification of DNA fromplant tissueContentsKit Contents4 Storage5 Intended Use6 Safety Information6 Quality Control6 Introduction7 Principle and procedure7 Description of protocols11 Equipment and Reagents to Be Supplied by User12 Important Notes14 Collection and storage of starting material14 Sample size14 Disruption and homogenization using the TissueRuptor15 Disruption and homogenization using the TissueLyser System15 Centrifugation(DNeasy96procedures)17 Lysate filtration with QIAshredder(DNeasy Mini and Maxi procedures)19 Elution19 DNA storage21 ProtocolsPurification of Total DNA from Plant Tissue(Mini Protocol)22 Purification of Total DNA from Plant Tissue(Maxi Protocol)26 Purification of Total DNA from Fresh Plant Tissue(DNeasy96Protocol)30 Purification of Total DNA from Frozen or Lyophilized Plant Tissue(DNeasy96Protocol)35 Troubleshooting Guide42 Appendix A:Determination of Yield,Purity,and Length of DNA46 Appendix B:Recovery and Cleaning of Beads and S-Blocks48 Ordering Information49DNeasy Plant Handbook10/20123Kit ContentsDNeasy Plant Mini and Maxi KitsDNeasy Plant Kits Mini(50)Mini(250)Maxi(6)Maxi(24) Catalog no.69104691066816168163 Number of preps50250624 DNeasy Mini Spin Columns(colorless)50250––DNeasy Maxi Spin Columns(colorless)––624 QIAshredder Mini SpinColumns(lilac)50250––QIAshredder Maxi SpinColumns(lilac)––624 Collection Tubes(2ml)50250––Collection Tubes(50ml)––624 Buffer AP140ml200ml40ml140ml Buffer P320ml2x50ml18ml50ml Buffer AW1(concentrate)*†2x19ml151ml25ml151ml Buffer AW2(concentrate)†17ml2x40ml26ml2x68ml Buffer AE2x12ml2x60ml15ml60ml RNase A(100mg/ml)220µl5x220µl110µl440µl Quick-Start Protocol1111*Contains a chaotropic salt.Not compatible with disinfectants containing bleach.See page6for safety information.†Buffer AW1and Buffer AW2are supplied as concentrates.Add ethanol(96–100%)according to the bottle label before use to obtain a working solution.4DNeasy Plant Handbook10/2012DNeasy96Plant KitDNeasy96Plant Kit(6)Catalog no.69181 Number of preps6x96 DNeasy96Plates6S-Blocks*2 Collection Microtubes,1.2ml(racked)12x96 Collection Microtube Caps4x(120x8) Elution Microtubes RS(racked)and caps6x96 AirPore Tape Sheets5x25 Buffer AP12x140ml Buffer P32x50ml Buffer AW1(concentrate)†‡151ml Buffer AW2(concentrate)‡2x81ml Buffer AE128ml RNase A(100mg/ml)2x440µl Reagent DX1ml96-Well-Plate Registers6Quick-Start Protocol1*Reusable;see Appendix B(page48)for cleaning instructions.†Contains a chaotropic salt.Not compatible with disinfectants containing bleach.See page6for safety information.‡Buffer AW1and Buffer AW2are supplied as concentrates.Add ethanol(96–100%)according to the bottle label before use to obtain a working solution.StorageDNeasy Maxi spin columns should be stored dry at2–8°C upon arrival and are stable for1year under these conditions.All other components of DNeasy Plant Kits,including RNase A stock solution,should be stored dry,at room temperature(15–25°C)and are stable for1year under these conditions.For storage longer than1year or if ambient temperatures often exceed25°C,we recommend keeping the RNase A stock solution at2–8°C.DNeasy Plant Handbook10/20125Intended UseDNeasy Plant Kits are intended for molecular biology applications.These products are not intended for the diagnosis,prevention,or treatment of a disease.All due care and attention should be exercised in the handling of the products.We recommend all users of QIAGEN®products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments,or to other applicable guidelines.Safety InformationWhen working with chemicals,always wear a suitable lab coat,disposable gloves,and protective goggles.For more information,please consult the appropriate safety data sheets (SDSs).These are available online in convenient and compact PDF format at /safety where you can find,view,and print the SDS for each QIAGEN kit and kit component.CAUTION:DO NOT add bleach or acidic solutions directly to wastecontaining Buffers AW1and AL.Buffer AW1contains guanidine hydrochloride,which can form highly reactive compounds when combined with bleach.If liquid containing this buffer 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 with1%(v/v)sodium hypochlorite.24-hour emergency informationEmergency medical information in English,French,and German can be obtained 24hours a day from:Poison Information Center Mainz,GermanyTel:+49-6131-19240Quality ControlIn accordance with QIAGEN’s ISO-certified Quality Management System,each lot of the DNeasy Plant Kits is tested against predetermined specifications to ensure consistent product quality.6DNeasy Plant Handbook10/2012IntroductionDNeasy Plant Kits provide a fast and easy way to purify DNA from plant and fungal tissue.Up to100mg of tissue can be processed using the DNeasy Plant Mini Kit or up to1g of tissue using the DNeasy Plant Maxi Kit.The DNeasy96Kit is designed for high-throughput DNA purification from50mg plant tissue per well(for some plant tissues,up to100mg per well can be used).Easy-to-use DNeasy Plant procedures provide pure total DNA(genomic,mitochondrial, and chloroplast)for reliable PCR and Southern blotting in less than1hour(DNeasy Plant Mini Kit)or2hours(DNeasy Plant Maxi Kit or DNeasy96Plant Kit).Purification requires no phenol or chloroform extraction or alcohol precipitation,and involves minimal handling.This makes DNeasy Mini and Maxi Kits highly suited for simultaneous processing of multiple samples.For higher-throughput applications,the DNeasy96 Plant Kit enables simultaneous processing of96or192samples.Purified DNA is eluted in low-salt buffer or water,ready for use in downstream applications.DNA purified using DNeasy Plant Kits is up to40kb in size,with fragments of20–25kb predominating.DNA of this length denatures completely in PCR and shows the highest amplification efficiency.Purified DNA has an A260/A280ratio of 1.7–1.9,indicating high purity of the DNA.The DNeasy membrane ensures complete removal of all inhibitors of PCR and other enzymatic reactions.DNA purified using DNeasy Plant Kits is highly suited for use in all downstream applications,including PCR,RAPD analysis,AFLP analysis,RFLP analysis,Southern blotting,microsatellite analysis,SNP genotyping,and quantitative, real-time PCR.Use of the TissueRuptor®or the TissueLyser,for rapid and convenient disruption of plant tissue samples,is recommended for the most efficient processing in DNeasy Plant procedures.Principle and procedureDisruption of plant tissueComplete and quick disruption of starting material is essential to ensure high DNA yields and to avoid DNA degradation.DNeasy Plant procedures are optimized for use with leaf tissues,but can also be used to purify DNA from other plant tissues and fungi, including seeds and spores.However,when using tissues other than leaves,the disruption method may require optimization to ensure maximum DNA yield and quality. DNeasy Plant Kits are compatible with all sample-disruption methods.Optimal results are obtained using the TissueRuptor homogenizer or the TissueLyser System.DNeasy Plant Handbook10/20127TissueRuptor homogenizerThe TissueRuptor is a handheld rotor–stator homogenizer designed for rapid,efficient, and flexible disruption of a variety of biological samples,including plant and animal tissues.Samples are simultaneously disrupted and homogenized by a rotating blade (rotor–stator homogenization).TissueRuptor disposable probes enable flexible sample disruption in a wide range of volumes and ing a separate disposable probe for each sample provides ease of use and prevents cross-contamination.Either fresh,frozen,or lyophilized plant tissue samples can be processed using the TissueRuptor.Tissues can be disrupted in liquid nitrogen or directly disrupted in lysis buffer without using liquid nitrogen,depending on the protocol and downstream application used.See“Disruption and homogenization using the TissueRuptor”, page15,for further details.TissueLyser SystemThe TissueLyser System provides convenient,simultaneous disruption and homogenization of multiple biological samples through high-speed shaking in plastic tubes with stainless steel,tungsten carbide,or glass beads.The TissueLyser II(cat.no.85300)provides medium-to high-throughput sample disruption of up to48or192samples.The TissueLyser LT(cat. no.85600)provides fast low-to medium-throughput sample disruption of up to12samples. Either fresh,frozen,or lyophilized plant tissue samples can be processed using the TissueLyser.Fresh material can be directly disrupted in lysis buffer without using liquid nitrogen.Alternatively,fresh or frozen samples can be disrupted after freezing in liquid nitrogen without lysis buffer.Lyophilized tissue can be disrupted without buffer at ambient temperature.See“Disruption and homogenization using the TissueLyser System”,page15,for further details.DNA purificationIn DNeasy Plant procedures(see flowcharts),plant material is first mechanically disrupted and then lysed by addition of lysis buffer and incubation.RNase A in the lysis buffer digests the RNA in the sample.After lysis,proteins and polysaccharides are salt-precipitated.In the DNeasy96procedures,cell debris and precipitates are removed by centrifugation.In DNeasy Mini and Maxi procedures,cell debris and precipitates are removed in a single step by a brief spin through the QIAshredder column,a unique filtration and homogenization unit.Binding buffer and ethanol are added to the cleared lysate to promote binding of the DNA to the DNeasy membrane.The sample is then applied to a DNeasy spin column or DNeasy96plate and centrifuged.DNA binds to the membrane while contaminants such as proteins and polysaccharides are efficiently removed by two wash steps.Pure DNA is eluted in a small volume of low-salt buffer or water.DNeasy purified DNA has A260/A280ratios of1.7–1.9,and absorbance scans show a symmetric peak at260nm confirming high purity.8DNeasy Plant Handbook10/2012DNeasy Plant Handbook 10/20129DNeasy Plant ProcedurePlant leaf tissueCollect plant tissuecleared and add buffer&washinto and Add ethanol &bind DNAGrind,lyse,&precipitatePlant tissueCentrifuge throughQIAshredderWash EluteReady-to-use DNA DNeasy 96Plant ProcedureThe DNeasy membrane combines the binding properties of a silica-based membrane with simple microspin technology or with the QIAGEN96-Well-Plate Centrifugation System(see ordering information,page50).DNA adsorbs to the DNeasy membrane in the presence of high concentrations of chaotropic salt,which remove water from hydrated molecules in solution.Buffer conditions in DNeasy Plant procedures are designed to enable specific adsorption of DNA to the silica membrane and optimal removal of carbohydrates,polyphenolics,and other plant metabolites.10DNeasy Plant Handbook10/2012Description of protocolsDifferent protocols in this handbook provide detailed instructions to use DNeasy Kits for purification of genomic DNA from plant tissue.The protocol“Purification of Total DNA from Plant Tissue(Mini Protocol)”,page22,is for use with the DNeasy Plant Mini Kit,with up to100mg(wet weight)fresh,frozen, or lyophilized plant tissue,following the specifications in Table1.The protocol“Purification of Total DNA from Plant Tissue(Maxi Protocol)”,page26,is for use with the DNeasy Plant Maxi Kit,with up to1g(wet weight)fresh,frozen,or lyophilized plant tissue,following the specifications in Table1.The“Purification of Total DNA from Fresh Plant Tissue(DNeasy96Protocol)”,page30, is for use with the DNeasy96Plant Kit,with fresh plant tissue,following the specifica-tions in Table1.This protocol is optimized for use with the TissueLyser System for effi-cient and convenient high-throughput disruption of plant tissue.The“Purification of Total DNA from Frozen or Lyophilized Plant Tissue(DNeasy96 Protocol)”,page35,is for use with the DNeasy96Plant Kit,with frozen or lyophilized plant tissue,following the specifications in Table1.This protocol is optimized for use with the TissueLyser System for efficient and convenient high-throughput disruption of plant tissue.Table1.Specifications for DNeasy Plant KitsDNeasy Plant Kit Mini Maxi96(amount/well) Maximum amount of100mg wet1g wet weight50mg wet weigth starting material*weight0.2g dry10mg dry weigth20mg dry weightweightDNA binding capacity†50µg500µg50µgMaximum loading volume700µl15ml1mlElution volume50–400µl500–2000µl100–200µl Typical yields3–30µg30–260µg1–15µg Preparation time<1hour<2hours<2hours(192samples)*Exceeding the recommended amount of starting material will reduce yield and purity.†Typically,the binding capacity will not be reached since the DNA content of the recommended amounts of starting material will not exceed30µg(Mini),300µg(Maxi),or50µg(96).Equipment and Reagents to Be Supplied by UserWhen working with chemicals,always wear a suitable lab coat,disposable gloves, and protective goggles.For more information,consult the appropriate safety data sheets(SDSs),available from the product supplier.For all protocolsEquipment for disruption and homogenizationWe recommend using the TissueRuptor or using the TissueLyser with the following accessories.Protocol TissueLyser accessoriesDNeasy Mini Adaptor Set2x24(cat.no.69982)Tungsten Carbide Beads,3mm(cat.no.69997)*1.5ml or2ml safe-lock microcentrifuge tubesDNeasy Maxi Grinding Jar Set,S.Steel(cat.no.69985)DNeasy96Adaptor Set2x96(cat.no.69984)Tungsten Carbide Beads,3mm(cat.no.69997)*Collection Microtubes(included with DNeasy96Plant Kit) *Stainless Steel Beads,5mm(cat.no.69989)can also be used.We recommend the use of tungsten carbide beads as these perform better and more consistently than stainless steel beads.Pipets and pipet tipsWater bath or heating block for heating at65°C and80°CVortexerEthanol(96–100%)†Liquid nitrogen(not required when processing lyophilized plant material nor when processing fresh plant material in the protocol“Purification of Total DNA from Fresh Plant Tissue(DNeasy96Protocol)”)For DNeasy Mini protocolMicrocentrifuge tubes(1.5ml)Microcentrifuge with rotor for2ml tubesIce†Do not use denatured alcohol,which contains other substances such as methanol or methylethylketone.For DNeasy Maxi protocol15ml and50ml centrifuge tubesThe use of disposable polypropylene tubes is recommended.Tubes used for the lysis step should be capable of withstanding the g-forces involved in centrifugation and should also be compatible with liquid nitrogen.Laboratory centrifuge(capable of3000–5000x g)equipped with a swing-out rotorAll centrifugation steps are carried out in a conventional laboratory centrifuge(e.g.,SIGMA®6-16series from SIGMA Laborzentrifugen GmbH,www.sigma-zentrifugen.de)*using a swinging bucket rotor.DNeasy Maxi spin columns and QIAshredder Maxi spin columns fit into the50ml centrifuge tubes provided.These tubes are compatible with almost all laboratory centrifuges and rotors.In the unlikely event that these tubes do not fit your rotor,the spin columns can also be used with any other commercially available50ml polypropylene or glass tubes.SpatulaIceFor DNeasy96protocolsCentrifuge4–16S or4–16KS with Plate Rotor2x96(see page17)Multichannel pipet with extended tipsFor efficient processing,we recommend the use of an electric multichannel pipet with a capacity of at least1ml per pipet tip.Options include the Matrix®Impact®cordless electronic multichannel pipet,which has a unique,adjustable tip-spacing system allowing the user to transfer liquid directly from sample tubes to96-well plates.We recommend using extended tips with a maximum volume of1250µl with the Matrix multichannel pipet(available from Matrix,cat.no.8255for tips with filters or8252for tips without filters).These multichannel pipets and pipet tips can be purchased from Matrix Technologies Corporation().*Reagent reservoirs for multichannel pipetsFreezer or cold room at–20°C*This is not a complete list of suppliers and does not include many important vendors of biological supplies.Important NotesCollection and storage of starting materialAfter harvesting,if plant tissue will not be used immediately,it should be frozen in liquid nitrogen.It can then be stored at–80°C for later processing.Ground tissue powder can also be stored at–80°C.Alternatively,tissue can be dried or lyophilized after harvesting to allow storage at room temperature(15–25°C).To ensure DNA quality, samples should be completely dried within24hours of collection.If possible,it is preferable to collect young materials(e.g.,leaves,needles)since they contain more cells per weight and therefore result in higher yields.In addition,young leaves and needles contain smaller amounts of polysaccharides and polyphenolics and are therefore easier to handle.When working with fungi,harvest mycelium directly from a culture dish or from liquid culture.For liquid culture,first pellet cells by centrifugation.Remove the supernatant completely before disruption and lysis.Fresh,frozen,or freeze-dried fungal material can be used.Sample sizeDNeasy Plant procedures are optimized for a maximum of100mg(Mini),1g(Maxi), or50mg(DNeasy96)*of wet-weight starting material.Table2provides guidelines for wet weights of leaf tissue.If using dried starting material the maximum amount which can be processed must be reduced by a factor of approximately5.Exceeding the recommended maximum amount of starting material will result in inefficient lysis, resulting in low yield and purity.*With some plant species it may be possible to increase the amount of starting material in the DNeasy96 procedure to increase DNA yield.For example,increasing the amount of wheat starting material to100mg increased the DNA yield by35%(27.4µg DNA compared with20.4µg DNA,after two100µl elutions).Note that DNA yields do not necessarily increase linearly with increased amounts of starting material.Furthermore for some plant species,impurities may be present in the purified DNA if the amount of starting material is increased.To find the optimum amount of starting material for a particular plant species,use a range of amounts(e.g.,50,75,and100mg),and determine the amount that provides the highest DNA yield and purity.Note:The maximum amount of starting material that can be used with the collection microtubes provided in the DNeasy96Plant Kit is50mg(wet weight).Other disruption vessels should be used for more than50mg starting material.Table2.Approximate wet weights of leaf materialSample Size Approximate wet weight(mg)Leaf(punch) 1.5cm diameter25–75Leaf(surface area)12cm2(e.g.,4x3cm)170–510DNA yields vary depending on genome size,ploidy,and age of sample.Yields typically range from3–30µg per100mg of wet-weight sample.Disruption and homogenization using the TissueRuptor Homogenization using the TissueRuptor system is carried out by first installing the adapter on the motor drive and placing a disposable probe into the adapter(see the TissueRuptor User Manual).Fresh or frozen samples can be disrupted after freezing in liquid nitrogen without Buffer AP1.Alternatively,fresh material can be directly disrupted in Buffer AP1without using liquid nitrogen,but this may cause shearing of high-molecular-weight DNA.We do not recommend disrupting frozen material in lysis buffer as this can result in low yields and degraded DNA.Especially hard tissues,such as roots or seeds,could cause the disposable probes to break and may not be well-suited for use with the TissueRuptor.These tissues can be disrupted using a mortar and pestle or the TissueLyser System.For optimal results,we recommend to keep the disruption time as short as possible. Disruption for more than1minute may lead to shearing of genomic DNA.Note:Disruption can be performed without lysis buffer by keeping the sample submerged in liquid nitrogen before and during disruption.In this case,Buffer AP1and RNase A stock solution(100mg/ml)need to be added to the sample immediately after disruption.Disruption and homogenization using the TissueLyser System Either fresh,frozen,or lyophilized plant tissue samples can be processed using the TissueLyser.Fresh material can be directly disrupted in lysis buffer without using liquid nitrogen.Alternatively,fresh or frozen samples can be disrupted after freezing in liquid nitrogen without lysis buffer.Lyophilized tissue can be disrupted without buffer at ambient temperature.Disruption of samples in lysis buffer yields DNA ideal for PCR, while disruption of samples in liquid nitrogen yields DNA of a higher molecular weight. We do not recommend disrupting frozen material in lysis buffer as this results in low yields and degraded DNA.Figure1.The TissueLyser II,TissueLyser LT,and TissueRuptor for sample disruption.IMPORTANT:When using the TissueLyser with frozen plant material,do not allow the adapter plates to come into contact with liquid nitrogen.Note:When using the TissueLyser Adapter Sets,samples on the inside of the adaptor rack move more slowly than samples on the outside.To prevent variation in sample homogenization,the adaptor sets should be removed from the TissueLyser and disassembled after the first disruption step.For the second disruption step,the adaptor sets should be reassembled so that the samples tubes that were outermost in the rack are now innermost.Rearranging the racks of collection microtubes in this way ensures that all samples are thoroughly and equally disrupted.IMPORTANT:Merely rotating the entire plate sandwich so that the QIAGEN logos are upside down when reinserted into the TissueLyser is not sufficient,since the same samples that were outermost during the initial disruption will remain outermost in the second disruption step.An instruction manual is provided with the TissueLyser.Read this manual carefully before using the TissueLyser.Do not use the TissueLyser with equipment other than that supplied.DNeasy Mini procedurePlant material and a tungsten carbide bead are added to a2ml safe-lock tube.Tubes are placed into the adaptor sets,which are fixed into the clamps of the TissueLyser. Disruption is performed in two1–2minute high-speed(20–30Hz)shaking steps.The beads are reusable(see Appendix B,page48,for cleaning instructions).For optimal operation,the TissueLyser should always be balanced.A balance can be provided by assembling a second adaptor using safe-lock tubes without samples or buffers,but containing the beads,and fixing this second adaptor into the empty clamp. DNeasy Maxi procedurePlant material is added to the grinding jars.The grinding balls are added,and the jars are fixed in the clamps of the TissueLyser.Disruption is performed in two1–2minute high-speed(20–30Hz)shaking steps.The grinding jars and balls are reusable(see Appendix B,page48,for cleaning instructions).For optimal operation,the TissueLyser should always be balanced.If using a single grinding jar,the balance should consist of a second grinding jar containing a stainless steel ball.DNeasy96proceduresFor DNeasy96procedures,plant material and a tungsten carbide bead are added to each of192collection microtubes in2racks.The racks are placed into the adaptor sets,which are fixed into the clamps of the TissueLyser.Disruption is performed in two 1–2minute high-speed(20–30Hz)shaking steps.The beads are reusable(see Appendix B,page48,for cleaning instructions).For optimal operation,the TissueLyser should always be balanced.A balance can be provided by assembling a second adaptor using a rack of collection microtubes without samples or buffers,but containing the beads,and fixing this second adaptor into the empty clamp.Centrifugation(DNeasy96procedures)Centrifuges4–16S or4–16KSDNeasy96spin protocols use a streamlined centrifugation procedure that enables purification of DNA from up to2x96samples in parallel for direct use in any downstream application.The DNeasy96Plant procedure requires use of the QIAGEN 96-Well-Plate Centrifugation System,comprising the Plate Rotor2x96and the table-top Centrifuge4–16S or the refrigerated table-top Centrifuge4–16KS(see page50for ordering information).In addition to the Plate Rotor2x96,a wide range of other rotors can be used with these centrifuges.Standard table-top centrifuges and microtiter plate rotors are not suitable for the DNeasy96protocol for2reasons:the microtiter plate buckets are either not deep enough for the complete DNeasy96package or they will not swing out properly,and furthermore,high g-forces(>5500x g)are required for optimal performance of the DNeasy96procedure.The speed limit of the Centrifuge4–16S and the Centrifuge 4–16KS(6000rpm;5796x g)is programmed so that the given g-force will not be exceeded.All centrifugation steps are performed at room temperature(15–25°C).IMPORTANT:Centrifuges must be properly maintained for optimal performance.It is particularly important that the buckets and rotor pins are routinely greased to prevent suboptimal running conditions that may lead to cracking of DNeasy96plates.For further information about QIAGEN Centrifuges and the Plate Rotor2x96,contact QIAGEN Technical Services or your local distributor(see back cover for contact information).Note:If the Centrifuge4–16KS is used,set the temperature to40°C for all centrifugation steps.Note:Use AirPore Tape Sheets(provided)to seal DNeasy96plates during all centrifugation steps to prevent cross-contamination between samples.Abbreviated instructions for using the Centrifuge4–16S and Centrifuge4–16KS Warning:Never run the centrifuge with empty plate carriers placed inside the buckets, that is,without the collection microtubes or DNeasy96plates and S-Blocks.If unsupported,the carriers will collapse under high g-forces.Therefore,remove the carriers during test runs.Standard microtiter plates may be centrifuged in the same carriers if the g-force does not exceed500x g.1.Switch on the centrifuge by pressing the main switch on the back.2.Select the rotor selection list in the display field by turning the knob.After pressingthe knob,turn the knob again to select the rotor/bucket combination “09100/09158”for the Plate Rotor2x96.Confirm entry by pressing the knob.Entering the rotor number automatically sets the time and speed limits for centrifugation for that particular rotor,thus eliminating the danger of the centrifuge over-speeding.3.Select“Speed”by turning the knob.Press the knob and by turning the knob again,set the speed to“6000”.Confirm entry by pressing the knob.The corresponding relative centrifugal force(RCF)is calculated from the rotor number and speed and appears automatically in the RCF field.It is also possible to enter the RCF value “5796x g”manually in the RCF field after selecting“RCF”in the same way. 4.Select“Time”by turning the knob.Press once and by turning the knob again,setthe time as recommended in the particular protocol step.Confirm entry by pressing the knob.5.For the Centrifuge4–16KS,set the temperature to40°C.6.Open the lid,place the96-well plates with the metal carriers in the buckets thenclose the lid.The start and lid keys light up.7.Push“Start”to start the centrifuge.When the centrifuge is running the lid key willnot be lit.Each run can be interrupted by pushing“Stop”.8.At the end of the run,the lid key will light up.Open the centrifuge lid by pressingthe lid key.Remove the plates.All preset parameters remain after a run has finished.。

质粒DNA中提方法（QIAfilter midi kit 25）适用于100 微克的高质粒或粘粒DNA，使用QIAfilter注射器样元件代替常规离心法以清除细菌裂解物。

低拷贝质粒，加入氯霉素放大后应视为高拷贝。

为质粒选择适当的培养体系。

在开始之前的注意事项■如果是低拷贝，最好增加裂解buffer的用量，以提高裂解效率，裂解缓冲卷，从而增加DNA 产量。

■可选：删除样品的步骤用符号表示☞为监测分析凝胶（见第34 页）的过程。

■加入buffer P3后，样品不再需要冰浴。

在开始之前做的东西■把提供的RNase A 添加到buffer P1 中。

一瓶RNase A (使用之前简要地离心）加入一瓶buffer P1 ，终浓度为100 ug/ml。

■检查buffer P2 是否由于低温存储产生SDS沉淀。

如有必要，37 ° C下使SDS溶解。

.■在4 °C预冷buffer P3。

■可选：使用前将提供的LyseBlue 试剂添加到buffer P1 混合。

每个buffer P1 瓶加入一小瓶LyseBlue （在使用之前简要离心），达到1: 1000 稀释。

LyseBlue 可以提供视觉识别从而防止常见处理错误，如细胞裂解导致效率低下和不完全沉淀的SDS、基因组DNA污染、细胞碎片等。

过程1.摇菌：新鲜划线的选择性平板（接种含有适当的选择性抗生素）挑一个单菌落放入2-5 ml LB 培养基中。

在37 ° C 摇菌约8 h （约300 rpm）。

使用4倍体系容积的试管或锥形瓶。

2.稀释选择性LB 培养基1/500 -1/1000。

若为高拷贝质粒，接种到25 毫升或● 100毫升的介质具有▲ 25—50 µ l 或● 100 – 200 µ l 起始培养液。

若为低拷贝质粒，接种▲ 50 — 100 毫升或● 250 毫升的介质具有▲ 100 – 200 µ l 或● 250-500 µ l 起始培养基。