C28025032.用于qRT-PCR的M-MLV第一链合成系统说明书
第一链cDNA合成试剂盒Transcriptor First Strand cDNA Synthesis Kit 中文说明书
第一链cDNA合成试剂盒Transcriptor First Strand cDNA Synthesis Kit中文说明书2013-12-07 13:20:16Transcriptor First Strand cDNA Synthesis Kit试剂盒包装与含量小瓶/瓶盖标签适用于a) 04 379012001b)04896866001c************1红色TranscriptorReverseTranscriptase（逆转录酶）a) 1瓶，25 μl (20 U/μl)b) 1瓶，50 μl (20 U/μl)c) 2瓶，各50 μl (20 U/ μl)•储存缓冲液：200 mM 磷酸钾，2 mM 二硫苏糖醇，0.2% Triton X-100(v/v)，50% 甘油(v/v)，pH 约为7.22无色Transcriptor RTReaction Buffer(5×)（逆转录缓冲液）a) 1瓶，1 mlb) 1瓶，1 mlc) 2瓶，各1 ml• 5×浓度：250 mM Tris/HCl，150 mM KCl，40 mM MgCl2，pH约为8.5(25°C)3无色ProtectorRNase Inhibitor（RNase抑制剂）a) 1瓶，50 μl(40 U/μl)b) 1瓶，100 μl(40 U/μl)c) 2瓶，各100 μl(40 U/μl)•储存缓冲液：20 mM Hepes-KOH，50 mM KCl，8 mM 二硫苏糖醇，50 % 甘油(v/v)，pH 约为7.6 (4°C)4黄色/紫色Deoxynuc-leo-tideMix（dNTP）a) 1瓶，100 μl(黄色瓶盖)b) 1瓶，200 μl(紫色瓶盖)c) 2瓶，各200 μl(紫色瓶盖)• dATP, dCTP, dGTP, dTTP各10 mM5蓝色Anchored-oligo(dT)18Primer（锚定oligo(dT)18引物）a) 1瓶，100 μl(50 μM)b) 1瓶，200 μl(50 μM)c) 2瓶，各200 μl(50 μM)6Random Hexamera) 1瓶，100 μl(600 μM)蓝色Primer（随机引物）b) 1瓶，200 μl(600 μM)c) 2瓶，各200 μl(600 μM)7绿色Control RNA（对照RNA）a) 1瓶，20 μl(50 ng/μl)•包含提取于永生细胞系(K562)的总RNA片段稳定溶液8绿色Control Primer Mix PBGD（对照基因引物）a) 1瓶，40 μl•5 μM 人类PBGD特异性正向与反向引物9(b和c为瓶7)无色Water, PCR-gradea) 1瓶，1 mlb) 2瓶，各1 mlc) 3瓶，各1 ml注意：货号为***********和***********的产品不含有control试剂(瓶7和瓶8)，因此瓶7即为Water, PCR-grade。
人cDNA第一链合成M-MLV酶联免疫分析
人cDNA第一链合成(M-MLV)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。
检测范围：96T20pg/ml-480pg/ml使用目的：本试剂盒用于测定人血清、血浆及相关液体样本中cDNA第一链合成(M-MLV)含量。
实验原理本试剂盒应用双抗体夹心法测定标本中人cDNA第一链合成(M-MLV)水平。
用纯化的人cDNA第一链合成(M-MLV)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入cDNA第一链合成(M-MLV)，再与HRP标记的cDNA第一链合成(M-MLV)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。
TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的cDNA第一链合成(M-MLV)呈正相关。
用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人cDNA第一链合成(M-MLV)浓度。
1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。
若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。
操作步骤1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。
2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。
在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。
加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。
3.温育：用封板膜封板后置37℃温育30分钟。
4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。
6.加酶：每孔加入酶标试剂50μl，空白孔除外。
7.温育：操作同3。
All-in-One qPCR Mix 产品说明书
All-in-One™qPCR MixFor universal quantitative real-time PCRCat.No.QP001(Old Cat.No.AOPR-0200,20μl×200reactions)Cat.No.QP002(Old Cat.No.AOPR-0600,20μl×600reactions)Cat.No.QP004(Old Cat.No.AOPR-1000,20μl×1000reactions)Cat.No.QP005(Old Cat.No.AOPR-4000,20μl×4000reactions)Performance optimized for All-In-One™qPCR Primers,All-In-One™miRNA qPCR Primers, miProfile™miRNA qPCR Arrays,ExProfile™Gene qPCR Arrays,All-In-One™First-Strand cDNA Synthesis Kit and All-In-One™miRNA First-Strand cDNA Synthesis KitUser ManualGeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850USA301-762-0888866-360-9531***********************©2016GeneCopoeia,Inc.USER MANUALAll-in-One TM qPCR MixI.DescriptionII.Related ProductsIII.Contents and StorageIV.PreparationV.ProcedureVI.ExampleVII.Trouble Shooting GuideVIII.Limited Use License and WarrantyI.DescriptionThe All-in-One™qPCR Mix provides fast and efficient SYBR®Green-based real-time quantitative PCR.The qPCR Mix uses a high-fidelity hot-start DNA polymerase,optimized reaction buffer and high-quality dNTPs to enable specific and sensitive amplification of even low-copy genes or miRNAs.The All-in-One TM qPCR Mix reduces experimental design time by providing a universal reaction condition that can be used with almost all primers and most real-time PCR instruments.II.Related ProductsGeneCopoeia offers comprehensive solutions for studying gene expression.A careful process of co-development ensures that they work well together and provide robust and reproducible results.Product DescriptionAll-in-One™First-Strand cDNASynthesis KitReverse transcribe mRNA into first–stand cDNAAll-in-One™qPCR PrimersValidated,gene-specific primers ensure specificity and sensitivity (human,mouse and rat)ExProfile™Gene qPCR Arrays High-throughput or focused group profiling of gene expression All-in-One™miRNA First-StrandcDNA Synthesis KitReverse transcribe miRNA into first–stand cDNAAll-in-One™miRNA qRT-PCR Detection Kits SYBR®Green-based detection kit accurately quantifies miRNA expressionAll-in-One™miRNA qPCR Primers Validated human,mouse,rat miRNA primers for robust,reproducible and reliable quantitation of miRNA activitymiProfile™miRNA qPCR Arrays High-throughput or focused group profiling of miRNA expression RNAzol®RT RNA Isolation Reagent Easy isolation of mRNA,microRNA or total RNAIII.Contents and StorageContents and storage recommendations for the All-in-One TM qPCR Mix are provided in the following table. Cat.Nos.QP001,QP002,QP004,and QP005Contents Quantity Storage temperature/conditions2×All-in-One TM qPCR Mix 2×1ml3×(2×1ml)5×(2×1ml)20×(2×1ml)–20°C(Stable for at least12months)Alternatively,the solution can also bestored at–80°C in aliquots.Avoidrepeated freezing/thawing.ROX Reference Dye (30μΜ)1×80µl3×80µl5×80µl20×80µl–20°C(Stable for at least12months)Alternatively,the solution can also bestored at–80°C in aliquots.Avoidrepeated freezing/thawing.IV.PreparationWearing a lab coat,disposable gloves and protective goggles are recommended when handling chemicals.IMPORTANT NOTES:1.When using the All-in-One qPCR Mix with miProfile miRNA qPCR Arrays and All-in-One miRNAFirst-Strand cDNA Synthesis Kit for miRNA expression profiling,please follow the miProfile miRNA qPCR array user manual for the complete instruction.2.Store the kit at–20°C.Avoid storage or leaving reagents at4°C or room temperature.Avoid lightexposure at all times.3.Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then brieflycentrifuge before use.4.Prepare the reaction mix with PCR grade water.5.Strictly follow standard procedures for PCR to avoid nucleic acid contamination and non-specificamplification.6.Read all procedures before setting up the PCR reactionV.Procedure1.Thaw the2×All-in-One TM qPCR Mix and ROX Reference Dye as needed.2.Prepare the PCR reaction mix on ice.See the example below.Reagent Volume Final concentration2×All-in-One TM qPCR Mix a10μl1×PCR forward primer(2µM)b2µl0.2µM cPCR reverse primer(2µM)2µl0.2µMTemplate d2μlROX Reference Dye e(30μΜ)ifneeded0.4-0.1μl600nM-150nMWater(double distilled)■Not using ROX Reference Dye4μl■Using ROX Reference Dye3.6-3.9μlTotal20μle the2×All-in-One TM qPCR Mix as half of the total reaction volume and adjust other reagentsaccordingly.If the total reaction volume is changed,maintain each component in the proper proportion. b.Primers are important considerations to ensure success with real-time PCR.All-in-One TM human,mouseand rat primer sets from GeneCopoeia have been validated to provide specific and sensitive amplification even with low copy number genes.For designing your own primers,you may wish to use Oligo primer analysis software(Molecular Biology Insights)or Primer Premier software(Premier Biosoft International).c.Primer concentration should be in the range of0.2to0.6µM.In general,a PCR reaction using0.2µMprimers produces good results.If the PCR efficiency is low,consider increasing primer concentration.However,keep in mind that non-specific PCR products may also increase with increased primer concentration.d.Generally,the amount of DNA template should be less than100ng.Because different templates containvarying copies of a target gene,it may be necessary to perform a gradient dilution to determine the optimal amount of DNA template to use.If reverse transcript cDNA is used as template,dilute before use.Do not add more than5%of the original cDNA solution volume to the total qPCR reaction solution.e.ROX Reference Dye is added only for qPCR instruments that require ROX for calibration.ROXReference Dye provides an internal reference to which the reporter-dye signal can be normalized during data analysis.Normalization is necessary to correct for fluorescence fluctuations due to changes in concentration or volume.Adjust the ROX Reference Dye to optimal concentration according to different qPCR instruments.Instrument ROX per20µl PCR Reaction Final Concentration BioRad iCycler，MyiQ，iQ5，CFX-96，CFX-384，Eppendorf Mastercyclerrealplex，Roche LightCycler480，LightCycler2.0None No ROXABI PRISM7000/7300/7700/7900HTand7900HTFast,ABI Step One,ABI Step One Plus0.4µl（0.2-0.4µl）600nM（300-600nM）ABI7500，7500Fast,ABI Viia7，Stratagene Mx3000P，Mx3005P,Mx4000，0.1µl（0.02-0.1µl）150nM（30-150nM）For other instruments which need calibration of ROX but have not been listed out in the table,please optimize the concentration of ROX according to the guide line of specific instrument.3.Mix the PCR reaction mix sufficiently and add to the PCR reaction tubes.4.Briefly centrifuge to make sure all the reagents are at the bottom of the reaction tubes.5.The following three-step method for programming the PCR reaction is recommended:Cycles Steps Temperature Time Detection 1Initial denaturation95°C10min No40Denaturation95°C10sec No Annealing55°C~60°C20sec No Extension72°C15sec YesNotesi.When using SYBR Green dye to monitor the qPCR reaction,a melting curve analysis should beperformed immediately at the end of cycling.(example adapted from the iQ5real-time PCRdetection system from Bio-Rad):Temperature range Heating rate Constant temperature Detection 72–95°C0.5°C/unit time6sec/unit time Yes25°C30sec NoThe conditions for your instrument may differ,consult the documentation of your qPCR instrument for instructions.ii.The DNA polymerase used in the2×All-in-One TM qPCR Mix is a special chemically modified hot-start enzyme.Incubation for10minutes at95°C will sufficiently activate the enzyme.iii.The actual annealing temperature should be adjusted around the primer melting temperature ranging from55°C~60°C.However,the optimal annealing temperature may be outside of thisrange.Adjust the temperature according to actual reaction conditionsiv.The optimal fragment length to use for amplification during real-time PCR is in the range of80-150bp.However,fragment lengths up to300bp are possible.v.The main condition for the above reaction are referred to in the iQ5qPCR instrument manual from Bio-Rad.If a qPCR instrument from another commercial source is used,please reference theinstrument manual and adjust the extention time and melting curve conditions accordingly.VI.ExampleObjective:The amplification efficiency and detection sensitivity of the2×All-in-One TM qPCR Mix are assessed by standard curves made by gradient dilution of plasmid DNA.The target fragment is102bp.Equipment:iQ5instrument(Bio-Rad Laboratories)Procedure:1.The plasmid is serially diluted to6concentrations ranging from105to1molecule/μl.2.PCR reaction mix preparation(on ice)Reagent components Volume2×All-in-One qPCR Mix10µlPCR forward primer(2µM)2µlPCR reverse primer(2µM)2µlddH201µlTotal15µl3.Mix the above reagents sufficiently.Aliquot to PCR tubes after a brief centrifugation.4.Add5μl of the diluted plasmid template to each PCR e5μl ddH2O as a negative control.5.Program the PCR reaction and corresponding reading conditions of the melting curve:Cycles Steps Temperature Time Detection1Initial denaturation95°C10min No45Denaturation95°C10sec No Annealing60°C20sec No Extension72°C15sec Yes Melting curve reading72°C~95°CHeating Rate0.5°C/6secYes Cooling25°C30sec No6.Analyze the amplification and corresponding melting curves after the qPCR experiment:Amplification curves of serially diluted plasmid DNA Peak values of amplified products in melting curves.7.Construct a standard curve using the Ct values from each amplification curve:Picture of a standard curve8.Conclusion:The peak values from the amplification and melting curves show that as low as5molecules can be detected when using plasmid DNA as a template and that there is only a single amplified product,showing that very high sensitivity can be attained using the All-in-One TM qPCR Mix.At the same time,high amplification efficiency is also shown by the good linear relationship among each concentration of serially diluted plasmid.VII.Trouble Shooting GuidePoor precision or failed qPCR reactions ∙Make sure the initial denature time was set as10min,sufficiently activating of the hot-start polymerase could avoid non-specific amplification and production of primer-dimers.∙The fluorescence detection temperature may not be appropriate.Adjust accordingly.∙The set up position for reaction samples in the real-time PCR instrument may not be right.Adjust accordingly.∙PCR cycle conditions,primer concentration and primer sequences may not be appropriate.Adjust the primer concentration and annealing temperature.If this does not work,redesign the primers.∙The template sample purity may not be adequate.Purify the template sample by phenol/chloroform extraction and ethanol precipitation.If the samples are reverse transcribed cDNA,set up the qPCR reaction with a diluted sample as other concentrated reagents in the RT reaction mixture may be interfering with the qPCR.∙Try to use 3.0%agarose gel electrophoresis to check the qPCR products.Check the purity of the primers by electrophoresis or use PAGE-purified primers if the bands are diffused.One may also use phenol/chloroform extraction and ethanol precipitation methods to treat the primers before the experiment.Abnormal meltingcurvesSignal in the blank(No Template Control)sample∙There may be contamination of the positive samples in the qPCR reaction system if the T m of the melting curve of the blank control is the same as the positive control.Eliminate sample application error first.If the situation still persists,replace the PCR grade water and/or primers and/or use a new2×All-in-One TM qPCR Mix.∙If the T m of the melting curve of the blank control is lower than the positive control,the qPCR reaction may have produced nonspecific amplification such as primer-dimers.Prepare the qPCR reaction mix on ice and increase the temperature of fluorescence detection.If this does not work,redesign the primers.Double peaks and multiple peaks in the melting curve of the positive control∙In the absence of other primers present in the reaction,double or multiple peaks in the melting curve of the positive control indicate that the qPCR reaction produced nonspecific amplification fragments.Prepare the qPCR reaction mix on ice;optimize the qPCR reaction conditions,for example,by increasing the annealing temperature, decreasing the primer concentration or increasing the fluorescence detection temperature(not more than the T m value of the expected product).If this does not work,redesign the forward primer.No peaks or abnormal peaks in the melting curve(or the amplification curves)of the positive control∙Adjust the ROX Dye to optimized concentration according to instrument.No signal(Ct)or late appearing signal ∙Not enough PCR cycles.For good sensitivity,one should generally set up more than35PCR cycles,but more than45cycles may result in too much background signal.∙The amount of template used may not be enough or the template may be e the highest concentration possible of diluted template samples to set up the qPCR.At the same time,avoid freezing and thawing the samples repeatedly.∙The amplification efficiency is low and the qPCR reaction conditions are not optimal.Redesign the primers and optimize the reaction conditions.VIII.Limited Use License and WarrantyLimited Use LicenseFollowing terms and conditions apply to use of all OmicsLink™ORF Expression Clones in all lentiviral vectors and Packaging Kit(theProduct).If the terms and conditions are not acceptable,the Product in its entirety must be returned to GeneCopoeia within5calendar days.A limited End-User license is granted to the purchaser of the Product.The Product shall be used by the purchaser for internal researchpurposes only.The Product is expressly not designed,intended,or warranted for use in humans or for therapeutic or diagnostic use.TheProduct must not be resold,repackaged or modified for resale,or used to manufacture commercial products without prior written consentfrom GeneCopoeia.This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetice of any part of the Product constitutes acceptance of the above terms.Limited WarrantyGeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet.If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications,GeneCopoeia will replace the Product.In the event a replacement cannot be provided,GeneCopoeia will provide the purchaser with a refund.This limited warranty shall not extend to anyone other than the original purchaser of the Product.Notice of nonconforming products must be made to GeneCopoeia within30days of receipt of the Product.GeneCopoeia’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price.GeneCopoeia’s liability does not extend to any damages arising from use or improper use of the Product,or losses associated with the use of additional materials or reagents.This limited warranty is the sole and exclusive warranty.GeneCopoeia does not provide any other warranties of any kind,expressed or implied,including the merchantability or fitness of the Product for a particular purpose.GeneCopoeia is committed to providing our customers with high-quality products.If you should have any questions or concerns about anyGeneCopoeia products,please contact us at301-762-0888.©2016,GeneCopoeia,Inc.GeneCopoeia,Inc.9620Medical Center Drive,#101Rockville,MD20850Tel:301-762-0888Fax:301-762-3888Email:***********************Web:GeneCopoeia Products are for Research Use Only Copyright©2016GeneCopoeia,Inc. Trademarks:GeneCopoeia™,All-in-One™,ExProfile™,miProfile™(GeneCopoeia Inc.);RNAzol®(Molecular Research Center,Inc.);SYBR®(Molecular Probes);iQ™5(Bio-Rad);ROX®(Invitrogen).QP001020216。
ThermoScientificRevertAidFirstStrandcDNASynthesisKitK说明书第一链cDNA合成试剂盒
RevertAid第一链cDNA Synthesis试剂盒#K1621, #K1622分析证明书质量控制#K1621Lot采用100 fg对照GAPDH RNA和对照引物进行RT-PCR反应，通过在1%琼脂糖上进行凝胶电泳和溴化乙锭染色显示得到足够量的496 bp的产物质量认证人：Jurgita Zilinskiene目录页码试剂盒组成 (2)存储条件 (2)产品说明 (2)注意事项 (3)操作步骤 (6)RT-PCR (6)合成cDNA用于克隆………..………………………………………7 实验对照……………………………………………………………….8 问题分析与解决 (10)** 一个单位的RiboLockRNase酶抑制剂抑制5ng RNA酶A 50%的活性。
存储条件试剂盒中所有组分应存储在-20°C。
对照用RNA可存储于-70°C以便长期使用。
产品说明RevertAid第一链cDNA合成试剂盒以mRNA或者总RNA为模板，高效合成第一链cDNA。
本试剂盒使用RevertAid M-MuLV反转录酶，它的RNA酶H的活性与AMV反转录酶相比较低。
该反转录酶可耐受42-50°C温度，合成的cDNA片段长度达13kb。
试剂盒中含有RiboLock 重组RNA酶抑制剂，防止RNA降解，可耐受55°C高温。
试剂盒同时含有oligo(dT)18和随机六聚体引物。
随机六聚体引物与模板非特异性地结合，以总RNA中任何RNA为模板合成cDNA。
oligo(dT)18选择性和RNA 3’po ly(A)配对结合，只以有poly(A)尾巴的mRNA为模板合成cDNA。
使用本试剂盒也可采用序列特异性引物。
合成的第一链cDNA能直接用作PCR或荧光定量PCR的模板，第二链cDNA的合成或线性RNA扩增，也可用于需要用带有放射性或非放射性核苷酸标记第一链cDNA的实验，比如将标记好的第一链cDNA 作为杂交实验中的探针或者用于微阵列分析。
普洛麦格RT-PCR系统TB手册说明书
TB本手册是产品K1002S，K1003S和K1005S的使用说明。
请废除以往版本。
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请访问该网站以确定您所使用的是该技术公告的最新版本。
Ⅰ. 描述 (1)Ⅱ. 产品组分和保存条件 (2)Ⅲ. 优化RT-PCR反应 (3)A. RNA模板 (3)B. 对照反应 (3)C. 核酸污染的避免 (4)D. Mg2+的浓度 (4)E. 引物设计 (4)F. 温度 (4)G. 孵育时间和循环数 (4)Ⅳ. 应用RT-PCR系统合成和分析RT-PCR产物 (5)A. 操作指南 (5)B. 结果分析 (6)C. 对照引物序列 (6)Ⅴ. 常见问题处理 (8)Ⅵ. 参考文献 (9)熟练用户的操作指南 (11)Ⅰ. 描述RT-PCR系统基于本公司生产的M-MLV RTase H-（Point Mutant）及Taq DNA Polymerase，对两者的缓冲液系统进行优化并相应调整酶量使之互相兼容，适用于各种RNA如total RNA、poly(A)+mRNA或转录合成的RNA及相应反转录引物（随机引物、oligo(dT)和专一序列引物）。
通过RT-PCR反应，本试剂盒可对低至1pg的RNA*进行扩增，如果靶序列在total RNA中含量极少或片段较长(>8kb)，则可能需要多至1ug的RNA 作为模板。
*RT-PCR的灵敏度受多种因素影响，如模板RNA的含量、纯度及完整性，所用引物不同等。
对total RNA和poly(A)+mRNA而言，起始RNA范围为1pg-1ug；对特定目标的RNA模板，起始范围为102-1010拷贝分子。
0871-622-5599 0851-859-9189配制合成cDNA反应组分M-MLV RTase H-和5X RT Buffer反应缓冲液，dNTP混合物，特异的上游引物，RNasin，RNA模板合成第1条cDNA链 42℃，60分钟配置PCR反应液第1条cDNA链和Taq酶，10X PCR反应液，dNTP混合物，特异的上下游引物，变性模板94℃，2分钟合成第2条链和扩增DNA25循环图1. RT-PCR系统操作概述Ⅱ产品组分产品名称目录号RT-PCR 系统 K1002S供实验室使用。
东盛生物 RT-PCR Kit（逆转录试剂盒） 说明书
0.6 μl RNasin , M-MLV ； 按下列条件进行反转录反应 42℃ 温浴 60 min（随即引物 37℃ 温浴 60 min） ； 5 终止反应 70℃ 温浴 5 min 终止反应，置冰上进行后续实验或-20℃ 保存。 6 用 RNase-free ddH2O 将反应体系稀释到 50 μl ，取 2-5 μl 进行 PCR 扩增反应。
20 次逆转录反应，R1012 可进行 100 次逆转
录反应（20 μl 标准 PCR 反应体系，每次使用 M-MLV 1 μl）。
M-MLV 储存液 20 mM Tris-HCl (pH 7.5) 200 mM NaCl 0.1 mM EDTA 1 mபைடு நூலகம் DTT 0.01% NP-40 50% glycerol 5xfirst-strand buffer 成分 250 mM Tris-HCl (pH 8.3 at 25 ℃) 375 mM KCl 15 mM MgCl2 50 mM DTT 保存条件 各组分均-20℃保存，避免反复冻融。
●
在逆转录反应中经常加入 RNase 抑制剂以增加 cDNA 合成的长度和产量。RNase 抑制剂要在第一链合成反应中，在缓冲
液和还原剂（如 DTT）存在的条件下加入，因为 cDNA 合成前的过程会使抑制剂变性，从而释放结合的可以降解 RNA 的 RNase。蛋白 RNase 抑制剂仅防止 RNase A，B，C 对 RNA 的降解，并不能防止皮肤上的 RNase，因此尽管使用了这些抑 制剂，也要小心不要从手指上引入 RNase。
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Script One-Step qRT-PCR Kit 产品说明书
95057 / IFU-034.1 REV 021Script ® One-Step qRT-PCR KitCat. No 95057-050 Size: 50 x 50-µL reactions Store at -25°C to -15°C95057-200200 x 50-µL reactionsDescriptionThe qScript One-Step qRT-PCR Kit is a convenient and highly sensitive solution for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan ® 5’-hydrolysis probes or molecular beacons on real-time quantitative PCR systems that do not require an internal reference dye. cDNA synthesis and PCR amplification are carried out in the same tube without opening between procedures. The system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity, enabling unbiased co-amplification of low copy transcripts in the presence of higher copy reference genes. The proprietary reaction buffer has been specifically formulated to maximize activities of both reverse transcriptase and Taq DNA polymerase while minimizing the potential for primer-dimer and other non-specific PCR artifacts. Highly specific amplification is crucial to successful qRT-PCR as non-specific product(s) can compete for amplification of the target sequence and impair PCR efficiency. A key component of this kit is AccuStart Taq DNA polymerase, which contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation at 95°C, the antibodies denature irreversibly, releasing fully active, unmodified Taq DNA polymerase. Instrument CompatibilityDifferent real-time PCR systems employ different strategies for the normalization of fluorescent signals and correction of well-to-well optical variations. It is critical to match the appropriate qPCR reagent to your specific instrument. The qScript One-Step qRT-PCR Kit does not contain an internal reference dye. Please consult the following table, or visit our web site at to find an optimized kit for your instrument platform(s).ReagentCat NosCompatible Real-Time PCR SystemsqScript One-Step qRT-PCR Kit, ROX 95058-050 95058-200 Applied Biosystems 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ qScript One-Step qRT-PCR Kit, Low ROX 95059-050 95059-200 Applied Biosystems 7500, 7500 Fast, ViiA™ 7 Stratagene MX4000™, MX3005P™, MX3000P™qScript One-Step qRT-PCR Kit95057-050 95057-200Bio-Rad CFX96™, CFX384™,iCycler iQ ®, iQ™5, MyiQ™ Opticon™, MiniOpticon™, Chromo4™Cepheid Smart Cycler ®; Qiagen/Corbett Rotor-Gene ® Eppendorf Mastercycler ® ep realplex Roche Applied Science LightCycler ® 480Components ReagentDescriptionqScript One-Step Reverse Transcriptase Optimized 50X formulation of recombinant MMLV reverse transcriptase for one-step RT-PCR.One-Step Master Mix (2X) 2X reaction buffer containing dNTPs, magnesium chloride, AccuStart Taq DNA polymerase, and stabilizers Nuclease-free waterStorage and StabilityStore components in a constant temperature freezer at -25°C to -15°C upon receipt. Repeated freezing and thawing of the reaction mix is not recommended.For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.95057 / IFU-034.1 REV 022Guidelines for One-Step qRT-PCR Thaw all components, except qScript One-Step RT, at room temperature. Mix vigorously, and then centrifuge to collect contents to thebottom of the tube before using. Place all components on ice after thawing. To maximize specificity, reactions should be assembled on ice. AccuStart Taq DNA polymerase is inactive prior to high temperatureactivation; however, qScript One-Step reverse transcriptase is active at lower temperatures. First-strand synthesis can be carried out between 42°C and 52°C. Optimal results are generally obtained with a 10-minute incubation at 48 – 50°C. We recommend a 5 minute incubation at 95°C to fully inactivate the RT prior to PCR cycling. Preparation of a reaction cocktail is recommended to reduce pipetting errors and maximize assay precision. Assemble the reactioncocktail with all required components except RNA template and dispense equal aliquots into each reaction tube. Add RNA to each reaction as the final step. Addition of sample as 5 to 10-µL volumes will improve assay precision. Suggested input quantities of template are: 1 pg to 1 µg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. After sealing each reaction, vortex gently to mix contents. Centrifuge briefly to collect components at the bottom of the reaction tube.Reaction Assembly Component Volume for 50-µL rxn. Final Concentration One-Step Master Mix (2X) 25 µl 1X Forward primer Variable 400 – 900 nM Reverse primer Variable 400 – 900 nM Probe Variable 50-200 nM Nuclease-free water Variable RNA template 5 – 10 µL VariableqScript One-Step RT 1 µL1X Final Volume (µL) 50 µLNote : For smaller reaction volumes (i.e. 25-µL reactions), scale all components proportionally. Reaction ProtocolIncubate complete reaction mix in a real-time thermal detection system as follows: cDNA Synthesis48 – 50°C, 10 min Initial denaturation95°C, 5 min PCR cycling (30 - 45 cycles) 95°C, 10 to 15s55 – 60°C, 30 to 60s (data collection step)Quality ControlKit components are free of contaminating DNase and RNase. The qScript One-Step qRT-PCR Kit is functionally tested in RT-qPCR. Kinetic analysis must demonstrate linear resolution over six orders of dynamic range (r 2 > 0.995) and a PCR efficiency > 90%Limited Label LicensesUse of this product signifies the agreement of any purchaser or user of the product to the following terms:1. The product may be used solely in accordance with the protocols provided with the product and this manual and for use with components contained in the kitonly. QIAGEN Beverly, Inc. grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this manual, and additional protocols available at . Some of these additional protocols have been provided by Quantabio product users. These protocols have not been thoroughly tested or optimized by QIAGEN Beverly, Inc.. QIAGEN Beverly, Inc. neither guarantees them nor warrants that they do not infringe the rights of third-parties.2. Other than expressly stated licenses, QIAGEN Beverly, Inc. makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4. QIAGEN Beverly, Inc. specifically disclaims any other licenses, expressed or implied other than those expressly stated.5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN Beverly, Inc. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.©2018 QIAGEN Beverly Inc. 100 Cummings Center Suite 407J Beverly, MA 01915 Quantabio brand products are manufactured by QIAGEN, Beverly Inc.Intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.qScriptis a registered trademark of QIAGEN Beverly, Inc.TaqMan is a registered trademark of Roche Molecular Systems, Inc. LightCycler is a registered Trademark of Roche. Applied Biosystems, StepOne, StepOnePlus, ViiA, and ROX are trademarks Life Technologies Corporation. Stratagene, MX3000P, MX3005P and MX4000 are trademarks of Agilent Technologies, Inc. Mastercycler is a trademark of Eppendorf. Rotor-Gene is a registered trademark of Qiagen GmbH. SmartCycler is a trademark of Cepheid. CFX96, CFX384, iCycler iQ, iQ5, MyiQ, Opticon, MiniOpticon and Chromo4 are trademarks of Bio-Rad Laboratories.。
上海英骏生物技术 M-MLV逆转录酶说明书
M-MLV逆转录酶说明书【货号】【规格】C28025-01110,000 单位C28025-014 50,000 单位C28025-021 200,000 单位浓度：200U/µl 保存于-20℃（勿除霜）【描述】莫洛尼氏鼠白血病毒逆转录酶（M-MLV RT）能以单链RNA或DNA为模板，在引物的引发下合成与模板互补的DNA链。
本逆转录酶是M-MLV的部分pol基因在大肠杆菌中的表达产物（1-3）。
使用M-MLV RT 可以合成长达7kb的第一链cDNA。
【组分】M-MLV逆转录酶5×第一链合成缓冲液[250mM Tris-HCl（pH8.3，室温），375 mM KCl，15mM MgCl2]0.1M DTT【保存缓冲液】20mM Tris-HCl（pH7.5），100mM NaCl，0.1mM EDTA，1mM DTT，0.01%（v/v）NP-40，50%（v/v）甘油【保存条件】将所有的组分保存于-20℃冰箱（勿除霜）。
【额外的组分】仅在使用前将5×第一链合成缓冲液和0.1M DTT在室温解冻，用完后迅速重新冰冻。
RNaseOUT™重组核酸酶抑制剂（40单位/μl）可从Invitrogen单独订购（货号：10777-019）。
【使用M-MLV逆转录酶进行第一链cDNA的合成】建立20μl 的反应体系可以用于逆转录1ng～5μg总RNA或1～500ng mRNA1．将以下组分加入无核酸酶的微量离心管中1μl oligo(dT)12-18(500μg/ml)，或50–250ng随机引物或2pmole基因特异性引物1ng-5μg总RNA或1ng-500ng mRNA1μl 10mM dNTP混合物（dATP，dGTP，dCTP和dTTP均为10mM，pH中性）加入灭菌蒸馏水至12μl2．混合物在65℃加热5分钟后，迅速置于冰上冷却。
短暂离心后，加入以下组分：4μl 5×第一链合成缓冲液2μl 0.1M DTT1μl RNaseOUT™核酸酶抑制剂（40单位/μl）（注意：如果起始RNA的量小于50ng，则必须加入RNaseOUT™）3．在离心管中轻轻将各种成分混合，并在37℃下孵育2分钟。
QCell-Pro One-Step qRT-PCR SuperMix Kit 使用说明书
QCell-Pro One-Step qRT-PCR SuperMix KitUser’s Manual and InstructionsProduct: QCell-Pro One-Step qRT-PCR SuperMix KitCatalog Number: K5055200, K5055400IntroductionqRT-PCR is a highly sensitive technique that is widely used for detection and quantification of RNA in tissues and cultured cells. Traditionally, quantitative PCR is performed in two steps: a first-strand cDNA synthesis step using reverse transcriptase, followed by a PCR step using a thermostable DNA polymerase. This Kit combines Reverse Transcriptase (MMLV-RTase) and RNase Inhibitor in a single mixture, with hotstart Taq DNA polymerase in a separate 2x reaction mix optimized for probe based qRT-PCR. Both cDNA synthesis and PCR are performed in a single tube using gene-specific primers and either cell lysate or RNA. A cell lysis buffer is provided in the kit to make cell lysates in less than 5 minutes at room temperature. The cell lysate can be used directly for qRT-PCR, bypassing RNA isolation procedure. The passive reference dye ROX is included in a separate tube to make the QCell-Pro One-Step qRT-PCR SuperMix adaptable for many real-time QPCR platforms.BioChain’s QRT-PCR SuperMix contains BioChain’s Taq polymerase with hot start capability. BioChain’s hot-start Taq polymerase improves PCR amplification reactions by decreasing non-specific amplification and preventing primer-dimer formation. This enzyme is activated after an initial 10 minutes heating at 95°C. And the real-time RT-PCR buffer is specially formulated to provide superior specificity and increase reverse transcription and amplification efficiency.216, 36, 6, 1Y=-3.304Log(x)+31.88, R2=0.999, Efficiency=100.8%Initial Quantity, Cell numberFigure 1. BioChain’s QCell-Pro One-Step QRT-PCR SuperMix provides sensitive detection down to a single cell. K562 cells were lysed according to the cell lysis protocol. 6-fold serial dilution of cell lsyate were prepared from 216 cells to 1 cell. GAPDH gene expression was detected using BioChain’s QCell-Pro qRT-PCR kit on Stratagene’s Mx3005P instrument. Efficiency as measured from standard curve was 100.8%, with a R2 value of 0.999.Features•Flexible and convenient – quantitating gene expression in cells (without isolating RNA) or RNA in one-step format•Save time – quick cell lysis procedure, and ready-to-use supermix reducing setup time and liquid handling steps•High Sensitivity – qRT-PCR from as low as 1 cell or 1 pg total RNA.• Versatile – compatible with a wide variety of cell linesApplications• Real-Time RT-PCR• Gene expression profiling• Gene knockdown verification• Array ValidationDescriptionComponents in this kit are prepared with pure chemicals according to our proprietary technology.QCell-Pro One-Step qRT-PCR SuperMix Kit provides a one-step, simple, robust, inexpensiveassay for detection and quantitative analysis of gene expression directly from cells or RNA withprobe based format.Quality Control1 kit of this lot has been tested for quantitating human GAPDH gene expression in a serial dilutionof cell lysate from 216 cells to 1 cell using Stratagene’s Mx3005P as a real time PCR instrument.Good linearity and great PCR efficiency is observed and consistent with the previous lot.ComponentsCatalog Number: K5055200: Reagents are sufficient for 200 assaysNo. Item AmountPart1. Cell Lysis Buffer 20 ml K5055200-11.25 ml x 2 K5055200-22. Pro qRT-PCR Reaction Mixture, 2x (containingHotstart Taq DNA polymerase)3. Reverse Transcriptase / RNase Inhibitor Mixture 100 µl K5055200-34. ROX Reference Dye 50 µl x 2 K5055200-45. Nuclease-Free PCR Grade Water 3 ml K5055200-5Catalog Number: K5055400: Reagents are sufficient for 400 assaysPartNo. Item Amount1. Cell Lysis Buffer 40 ml K5055400-11.25 ml x 4 K5055400-22. Pro qRT-PCR Reaction Mixture, 2x (containingHotstart Taq DNA Polymerase)3. Reverse Transcriptase / RNase Inhibitor Mixture 100 µl x 2 K5055400-34. ROX Reference Dye 50 µl x 4 K5055400-45. Nuclease-Free PCR Grade Water 3 ml x 2 K5055400-5Reagents and Equipments Required but not Supplied in this Kit:1. PBS (Ca2+, Mg2+ free)2. Spectrofluorometric thermal cyclerStorage and StabilityUpon receipt, store all components at -20 ºC in a constant temperature freezer. Avoid repeated freeze/thaw cycles. When stored under these conditions the supermix is stable for one year after ship date. The ROX reference dye is light sensitive and should be kept away from light whenever possible.ProtocolPrimer and Probe DesignDesign QPCR primers to generate amplicons of ≤150 bp. Since the cell lysate contains genomic DNA, the primers and probe should be designed to amplify cDNA but minimize amplification of genomic DNA. It is useful to choose primers or probe that span an exon-exon boundary in the target mRNA, or choose primers that flank a large intron. If possible, design primers and probe to avoid regions of secondary structure in the mRNA. Since reverse transcription and PCR are performed in one-step, we recommend to use the reverse PCR primer as the gene specific primer for reverse transcription.Recommended Control ReactionsNo Template Control (NTC): no-template control reactions are recommended in each experiment to screen for contamination of reagents or false amplification.No-RT Control: no-RT control reactions are recommended for each experimental sample by omitting reverse transcriptase from the reaction. The no-RT control should generate no signal if the primers are specific for the cDNA and does not amplify genomic DNA.Use of the ROX Reference DyeROX reference dye is included in this kit and may be added to compensate for non-PCR related variations in fluorescence. Addition of the reference dye is optional. Optimizing the ROX dye concentration within the qPCR reaction is an important aspect of setup. Too much ROX in the qPCR reaction will reduce background but also makes a low target signal difficult to distinguish from background. Conversely, too little ROX can increase background, meaning that low or weak target signals can be lost. For instruments that allow excitation at ~584 nm (such as Stratagene’s Mx instrument and ABI 7500), firstly 1:10 dilute the ROX reference dye provided in the kit, then begin optimization using 0.5 µl diluted ROX reference dye in 25 µl qRT-PCR reaction. For instruments that do not allow excitation near 584 nm (such as ABI PRISM®/GENEAmp® 5700 instruments), begin optimization using 0.5 µl undiluted ROX reference dye in 25 µl qRT-PCR reaction.Reagent Preparation and StorageThaw the tube containing 2x qRT-PCR Reaction Mixture on ice and store it on ice while setting up the reactions.1. If the ROX reference dye will be included in the reaction, keep all solutions containing theROX protected from light.2. Due to the sensitivity of quantitative PCR, results can be easily affected by pipettingerrors. Always prepare a master mix of qRT-PCR supermix containing the primers andthe reference dye (if reference dye is used). Individual pipetting of replicate samples isnot recommended.Cell Lysis ProcedureThe lysis buffer can be used to prepare lysates from a variety of mammalian culture cells.Lysates may be prepared with the maximum cell density (104 cells /µl). When used for qRT-PCR,the lysate may be diluted in the cell lysis buffer prior to adding to the qRT-PCR reaction. Highconcentration of either cellular materials or lysis buffer may inhibit qRT-PCR reaction, so the totalamount of cell lysate added to the qRT-PCR reaction should not exceed 1/10 volume of thereaction. And the number of cells added to the 25 µl qRT-PCR reaction should be <= 2,000. Thisis a general guideline. For some cells lines, 2,000 cells may inhibit the qRT-PCR reaction. Prior tothe experiment, perform a pilot standard curve to determine the maximum number of the cellsthat may be added to the qRT-PCR reaction, and determine the cell number range that give linearamplification of the specific target under your specific reaction conditions.1. Harvest cells using the method appropriate to the properties of the cell line. For adherentcells, trypsinize the cells using standard techniques. Count the cell.2. Pelleting the cells by centrifuging at 200 – 300x g for 5 min. Carefully remove thesupernatant by aspiration.3. Wash the pellet once with ice-cold PBS. Pelleting the cells by centrifuging at 200 – 300xg for 5 min. Carefully remove the supernatant by aspiration. Keep the pellet on ice.4. Add appropriate volume of Cell Lysis Buffer to the cell pellet. Vortexing for 1 minute tolyse the cells.5. Analyze the lysate by qRT-PCR. RNAs in the lysate are stable at 4°C for up to 4 hr.QRT-PCR setup and cycling1. Prepare the following RT-PCR reaction mixture. (First make the master mix without thetemplate. After making the master mix, gently mix the reaction without creating bubbles,aliquot and then add 1 – 2.5 µl of template to each experimental reaction)per reaction: 25 µlConcentrationFinal Regents VolumePro QRT-PCR Reaction Mixture (2x) 12.5 µl 1xReverse Transcriptase / RNase0.5 µlInhibitor MixturePCR forward primer X µl 150 – 200 nMPCR reverse primer X µl 150 – 200 nMProbe X µl 150 – 500 nMROX Reference Dye a 0.5 µlTemplate (cell lysate or RNA)b 1 – 2.5 µlNuclease-free PCR grade water Add up to 25 µla See page 4: Use of the ROX Reference Dyeb If cell lysate is used as the template, the volume of cell lysate should not exceed 1/10volume of the qRT-PCR reaction. If RNA is used as the template, it is recommended touse RNA template in less than 1 µg.2. Gently mix the reactions without creating bubbles since bubbles interfere withfluorescence detection. Then centrifuge the reactions briefly.3. Place the reactions in the instrument and run the appropriate RT-PCR program. Try thefollowing protocol first, and optimize the reaction conditions if needed.PCR program for RT-PCR:Cycles Temperature Time Detection Remarkmin OFF1 42°C 151 95°C 10 min. OFF This step inactivates thereverse transcriptase andactivates the hotstart Taq DNApolymerase. 10 minutesincubation is required to fullyactivate hotstart Taq DNApolymerase.sec OFF4095°C 15sec ON50-60°C a 15sec OFF72°C 30a. Set an appropriate annealing temperature for the primer set used.4. Dissociation Program for all PCR productsFollow manufacturer’s guidelines for setting up dissociation depending on theinstrument’s software version.Related ProductsQCell-Eva One-Step qRT-PCR SuperMix Kit (Cat# K5054200, K5054400), Eva QPCR SuperMix (Cat# K5052200, K5052400), Pro QPCR SuperMix (Cat# K5053200, K5053400), dNTP set for PCR (Cat# K6011100), PCR mix (Cat# 5051100), PCR Optimization Kit (K5051100), Taq Polymerase (Cat#7051200), RNA, PCR ready cDNA, and PCR ready genomic DNA.References1. Higuchi R, Dollinger G, Walsh P S and Griffith R (1992): Simultaneous amplification anddetection of specific DNA sequences. BioTechnology 10:413-417.2. Higuchi R, Fockler C, Dollinger G and Watson R (1993): Kinetic PCR analysis: real-timemonitoring of DNA amplification reactions. BioTechnology 11:1026-10303. Bustin, S A (2000): Absolute quantification of mRNA using real-time reverse transcriptionpolymerase chain reaction assays. Journal of Molecular Endocrinology 25:169-193.。
美链 eva qpcr supermix kit 使用说明书
User’s Manual and InstructionsEva QPCR SuperMix KitCatalog Number: K5052200, K5052400IntroductionReal–time or quantitative PCR (QPCR) allows quantification of DNA, cDNA, or RNA templates. QPCR is based on the detection of a fluorescent reporter molecule that increases as PCR products accumulate with each cycle of amplification. In BioChain’s Eva QPCR SuperMix, a superior green fluorescence DNA-binding dye is used for real-time detection and quantitation of DNA. The Eva QPCR SuperMix is a ready-to-use, 2x-concentrated master mix that contains all the reagents (except primers and templates) needed for running quantitative, real-time DNA detection assays, in the double-stranded DNA dye detection format. The passive reference dye ROX is included in a separate tube to make the Eva Supermix adaptable for many real-time QPCR platforms. A pair of human beta-actin primers is also included in the kit as a control. BioChain’s QPCR SuperMix contains BioChain’s Taq polymerase with hot start capability. BioChain’s hot-start Taq polymerase improves PCR amplification reactions by decreasing non-specific amplification and preventing primer-dimer formation. This enzyme is activated after an initial seven to ten minutes heating at 95°C. And the real-time PCR buffer is specially formulated to provide superior specificity and increase amplification efficiency. This SuperMix can amplify and detect a broad range of DNA or cDNA targets, including those are GC- or AT-rich.Eva DyeEva Dye binds double–stranded DNA. Detection is monitored by measuring the increase in fluorescence intensity throughout the cycle. Eva Dye has higher affinity to double-stranded DNA than SYBR Green dye and shows stronger fluorescence intensity than SYBR Green upon binding to DNA. Eva Dye is more stable than SYBR Green and the absorption and emission spectra of Eva Dye are similar to SYBR Green Dye or FAM, so the same optical setting for SYBR Green Dye or FAM can also be used for Eva Dye.Y=-3.289Log(x)+37.56, R 2=0.999, Efficiency=101.4%Figure 1. BioChain Eva QPCR SuperMix amplifies over a broad dynamic range. 2 x 101 to 2 x 108 copies of plasmid containing cDNA of human beta-actin gene were amplified in 25 µl reactions. Highly reproducible triplicates demonstrated good linearity of 0.999 and excellent PCR efficiency of 101.4% over an 8-order of dynamic range. BioChain’s Eva SuperMix has high sensitivity, detecting as few as 20 copies of target DNA within the linear range.Figure 2. Dissociation Curve of PCR products amplifies over a broad dynamic range. 2 x 101 to 2 x 108copies of plasmid containing cDNA of human -actin gene were amplified in 25-µl reactions.1x102 1x103 1x104 1x105 1x106 1x107 1x108Initial Quantity, Copy NumberFeatures∙Convenient - All reaction components are supplied for quick and easy set up∙Save time - Ready-to-use SuperMix reduces setup time and liquid handling steps∙Wide dynamic range: good linearity and excellent PCR efficiency over an 8 orders of dynamic range∙High Sensitivity - detect as low as 20 copies of DNA.∙Amplify and detect a broad range of DNA or cDNA targets- including those that are GC- or AT-rich∙Flexible – Compatible with most of the real-time PCR instruments.Applications∙ Real-Time PCR∙Gene expression profiling∙Gene knockdown verification∙ Array validationDescriptionComponents in this kit are prepared with pure chemicals according to our proprietary technology. BioChain’s QPCR SuperMix is a 2x concentration of premix reagent including Hotstart DNA polymerase and Eva Dye and specially formulated real time buffer designed for real-time PCR with intercalator format.Quality Control1 kit of this lot has been tested for amplifying plasmid containing human β-actin cDNA (amplified fragment: 202 bp) over an 8 orders of dynamic range using Stratagene’s Mx3005P as a real time PCR instrument. Good linearity and great PCR efficiency is observed and consistent with the previous lot.ComponentsEva QPCR SuperMix Kit:Catalog Number: K5052200: Reagents are sufficient for 200 assaysItem Amount Part No.1. Eva QPCR SuperMix 1.25 ml x 2 K5052200-12. ROX reference Dye 50 μl x 2 K5052200-23. human β-actin control F/R primer pair (25x) 50 μl K5052200-3 Catalog Number: K5052400: Reagents are sufficient for 400 assaysItem Amount Part No.1. Eva QPCR SuperMix 1.25 ml x 4 K5052400-12. ROX reference Dye 50 μl x 4 K5052400-23. human β-actin control F/R primer pair (25x) 50 μl K5052400-3Reagents and Equipments Required but not Supplied in this Kit:1. Nuclease-free PCR-grade water2. Spectrofluorometric thermal cyclerStorage and StabilityUpon receipt, store all components at -20 ºC in a constant temperature freezer. Avoid repeated freeze/thaw cycles. When stored under these conditions the supermix is stable for 6 months after ship date. You may aliquot the supermix and store a portion at 4°C for ready use. The thawed Eva QPCR SuperMix is stable at least for 3 months at 4ºC. The Eva Dye and the ROX reference dye are light sensitive and should be kept away from light whenever possible.Protocol(Using Stratagene’s Mx3000P TM/Mx4000®, and ABI PRISM®/GENEAmp® 5700Real-time PCR Instrument)Use of the ROX Reference DyeROX reference dye is included in this kit and may be added to compensate for non-PCR relatedvariations in fluorescence. Addition of the reference dye is optional. Optimizing the ROX dyeconcentration within the qPCR reaction is an important aspect of setup. Too much ROX in theqPCR reaction will reduce background but also makes a low target signal difficult to distinguishfrom background. Conversely, too little ROX can increase background, meaning that low orweak target signals can be lost. For instruments that allow excitation at ~584 nm (such asStratagene’s Mx instrument and ABI 7500), firstly 1:10 dilute the ROX reference dye provided inthe kit, then begin optimization using 0.5 μl diluted ROX reference dye in 25 μl qRT-PCRreaction. For instruments that do not allow excitation near 584 nm (such as ABIPRISM®/GENEAmp® 5700 instrument), begin optimization using 0.5 μl undiluted ROXreference dye in 25 μl qRT-PCR reaction.Reagent Preparation and StorageThaw the tube containing Eva QPCR SuperMix on ice and store it on ice while setting up thereactions. After initial thawing, aliquot the supermix and store a portion at 4ºC for ready use.Avoid direct light in preparation of the PCR reaction mixture because Eva Dye is light sensitive.1. If the ROX reference dye will be included in the reaction, keep all solutions containingthe ROX protected from light.2. (Optional) Set up a no-template control to screen for contamination of reagents or falseamplification.3. Due to the sensitivity of quantitative PCR, results can be easily affected by pipettingerrors. Always prepare a master mix of Eva SuperMix containing the primers and thereference dye (if reference dye is used). Individual pipetting of replicate samples is notrecommended.Real-time PCR Cycling Programs4. Prepare the following PCR reaction mixture. (First make the master mix without thetemplate. After making the master mix, gently mix the reaction without creating thebubbles, aliquot and then add 2 μl of template to each experimental reaction)per reaction: 25 μlConcentration Regents VolumeFinal Eva QPCR SuperMix (2x) 12.5 μl 1xPCR forward primer X μl 100-200 nMPCR reverse primer X μl 100-200 nMReference Dye ROX a 0.5 μlTemplate b 2 μlNuclease-free PCR grade water Add up to 25 μlaSee page 5: Use of the ROX Reference Dye bFinal template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series. It is recommended to use DNA template in less than 100 ng.5. Gently mix the reactions without creating bubbles since bubbles interfere withfluorescence detection. Then centrifuge the reactions briefly.6. Place the reactions in the instrument and run the appropriate PCR program. Try thefollowing protocol firstly, and optimize the reaction condition if needed.PCR program for amplification:Cycles Temperature Time Detection Remark1 95°C 7-10 min. OFF This step will activate the Taqpolymerase.4095°C 30 sec OFF Set the instrument to detectand report fluorescence eitherat the annealing step or the extension step of each cycle.55-65°C a1 min ON 72°C 30 sec to 1.5 min bOFF172°C 3 min OFF This step can be omitted if theamplicon size is <300 bp.a. Set an appropriate annealing temperature for the primer set used.b. Set the extension time to 1-1.5 min if the amplicon size is > 400 bp.2. Dissociation Program for all PCR productsFollow manufacturer’s guidelines for setting up dissociation depending on the instrument’s software version.PCR Setup and Cycling Program for human β-actin control primer set (amplicon size = 202 bp)1. Prepare the following PCR reaction mixture. (First make the master mix without thetemplate. After making the master mix, gently mix the reaction without creating the bubbles, aliquot and then add 2 μl of template to each experimental reaction).per reaction: 25 μlRegents Volume Eva QPCR SuperMix (2x) 12.5 μl Human β-actin primer set (25x) 1 μlReference Dye ROX a0.5 μlTemplate b2 μl Nuclease-free PCR grade water Add up to 25 μlaSee page 5: Use of the ROX Reference Dye bFinal template concentration varies depending on the copy number of target present in the template solution. Optimal amount should be determined by preparing the dilution series2. PCR program for amplification of human β-actin amplicon.Cycles Temperature Time Detection1 95°C 10 min. OFFsec OFF40 95°C 3055-65°C 1 min ON72°C 30sec OFF3. Dissociation Program: Follow manufacturer’s guidelines for setting up dissociationdepending on the instrument’s software version.Related ProductsPro QPCR SuperMix (Cat# K5053200, K5053400), dNTP set for PCR (Cat# K6011100), PCR mix (Cat# 5051100), PCR Optimization Kit (K5051100), Taq Polymerase (Cat#7051200), RNA, PCR ready cDNA, and PCR ready genomic DNA.References1. Biotium, Inc. at /product/product_info/allcolor.pdf2. Brown, Katharine, et al. "SIRT3 reverses aging-associated degeneration." Cellreports 3.2 (2013): 319-327.3. Gray, Nora E., et al. "Angiopoietin-like 4 (Angptl4) protein is a physiological mediator ofintracellular lipolysis in murine adipocytes." Journal of Biological Chemistry 287.11(2012): 8444-8456.。
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用于qRT-PCR的M-MLV第一链合成系统
货号:C28025-032* 规格：50次反应
储存条件：–20℃ *注 该产品亦包含在以下两步法qRT-PCR试剂盒中：C11727-050、C11729-050、C11740-050、C11744-050 产品描述
用于qRT-PCR的M-MLV第一链合成系统适用于通过纯化的poly(A)+或总RNA合成第一链cDNA。
起始物质可以是1 ng至5 µg总RNA或1至500 ng mRNA。
首先，通过总RNA或poly(A)+选择性RNA，采用oligo(dT)、随机引物或基因特异性引物，进行cDNA合成。
下一步，利用qPCR SuperMix在单独的试管内进行qPCR反应。
两步法qRT-PCR试剂盒
用于qRT-PCR的M-MLV第一链合成系统包含在以下两步法qRT-PCR试剂盒中：
试剂盒名称反应次数货号
带有ROX的两步法M-MLV Platinum® SYBR® Green qPCR SuperMix-UDG
100次qPCR反应 C11744-050
100次qPCR反应
两步法M-MLV Platinum® SYBR® Green qPCR SuperMix-UDG 100次qPCR反应 C11740-050
带有ROX的两步法M-MLV Platinum®定量PCR SuperMix-UDG 100次qPCR反应 C11729-050
两步法M-MLV Platinum®定量PCR SuperMix-UDG 100次qPCR反应 C11727-050
组分
系统组分 数量
Oligo(dT)20 (50 µM) 50 µl
随机六聚物（50 ng/µl） 250 µl
5X第一链合成缓冲液（含15 mM MgCl2） 1 ml
0.1 M DTT 500 µl
10 mM dNTP混合物 50 µl
M-MLV 逆转录酶 50 µl
简要流程
产品编号：C28025032.pps 修订日期：2008年12月6日
问题解决指南
问题 可能原因
一般解决办法
第一链cDNA 合成步骤出错 以附带的总HeLa RNA 为对照，验证第一链反应的效率。
RNA 酶污染
确保无菌条件，避免RNA 酶污染。
在第一链反应中使用RNaseOUT™重组RNA 酶抑制剂。
多糖与RNA 发生共沉淀
按照Sambrook 等人（(1989).分子克隆实验指南，冷泉港实验室出版社）所述方法，用氯化锂沉淀RNA，以除去多糖。
目标mRNA 含有很强的转录停顿位点在第一链反应中用随机六聚物代替oligo(dT)。
使用距离目标cDNA 3´端更近的PCR 引物。
PCR 反应中所用第一链产物过少 最多可使用10 µl 的第一链反应产物。
第一链合成时使用了GSP （基因特异性引物）。
更换GSP 或者换用oligo(dT)。
确保GSP 位于反义序列上。
无qPCR 反应产物
体系内存在反转录抑制剂
第一链反应前用乙醇对mRNA 样品进行沉淀，以除去反转录抑制剂。
用70% (v/v)的乙醇漂洗mRNA 沉淀。
注：反转录抑制剂包括十二烷基硫酸钠(SDS)、EDTA、胍盐、甲酰胺、焦磷酸钠和精脒。
反应体系被基因组DNA 污染
按照DNA 酶I 说明文档所述方法，用扩增级DNA 酶I （货号：18068-015）对RNA 进行预处理。
设计引物时确保退火过程中引物与mRNA 上内含子两侧的外显子序列或者相邻两个外显子交界处的序列配对结合，以便区分扩增的cDNA 与污染的基因组DNA。
要检测产物是否来自DNA，可用RT 阴性对照进行验证。
引物发生非特异性退火 改变退火条件。
为每一对模板和引物组合优化镁离子浓度。
凝胶分析时发现杂带
引物形成二聚体
设计3´端没有互补序列的引物。
参考文献
Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor.
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