EnzymoPure™M-MuLV反转录酶(RTM-MuLVReverse Transcriptase)是一种经过改造和优化的M-MuLV反转录酶。和普通的M-MuLV反转录酶相同，RTM-MuLV反转录酶也是一种依赖于RNA或DNA模板的DNA聚合酶，可以以RNA或DNA为模板在引物存在的情况下完成互补DNA链的合成；同时具有RibonucleaseH(RNaseH)酶活性，可以选择性剪切RNA和DNA杂合双链中的RNA，但不剪切单链RNA或双链RNA。
特点：
M-MuLV反转录酶可以合成长达13kb的cDNA，而普通的M-MuLV反转录酶仅能合成长达9kb的cDNA；M-MuLV反转录酶的最适反应温度为42℃并且在50℃时仍然有很高活性，可以避免普通的M-MuLV反转录酶在37℃反应时的一些不足。
用途：
M-MuLV反转录酶常用于在获得总RNA或mRNA后cDNA第一条链的合成。M-MuLV反转录酶合成的cDNA第一条链后续可以用于PCR、real-timePCR、cDNA第二条链的合成等。M-MuLV反转录酶也可以用于DNA探针的标记，以及通过引物延伸(primerextention)来分析RNA。
来源：
M-MuLV反转录酶由大肠杆菌表达，表达的基因为经过突变优化的编码MoloneyMurineLeukemiaVirusreversetranscriptase的pol基因片段。
酶储存溶液：
50 mM Tris, pH 8.3, 100mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% Triton X-100 and 50% glycerol。Reaction Buffer(5X)：250 mM Tris, pH 8.3 at 25℃, 250 mM KCl, 20 mM MgCl2, 50 mM DTT。
失活或抑制：
70℃孵育10分钟可以导致M-MuLV反转录酶失活；EDTA、EGTA等螯合剂、无机磷酸盐或焦磷酸盐以及聚氨(polyamine)对M-MuLV反转录酶有抑制作用。本产品用于体积为20微升的反转录体系时足够进行10次反转录反应。
注意事项：
对于GC含量比较高的RNA的反转录，试剂盒的使用说明中给予了特别说明，请予以关注。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用说明：
1.cDNA第一条链的合成(First-strand cDNA Synthesi)：a.参考如下表格设置反转录反应：模板(后面3种任选一种)Total RNA0.1-5μg或Poly(A) RNA/mRNA10-500ng或Specific RNA0.01pg-500ng引物(后面3种任选一种)Oligo(dT)18 0.5μg (或100pmol)random hexamer0.2μg (或100pmol)Gene specific primer15-25pmolDEPC-treated Water－To 13.7μl*Reaction Buffer (5X)－4μlRNase Inhibitor－0.5μl**dNTP Mix (25 mM each)－0.8μl ***RT M-MuLV反转录酶－1μl总体积20μl*To13.7μl表示加入DEPC-treated Water至最终体积为13.7μl。注意：对于GC含量比较高的RNA模板的反转录反应， 加入DEPC-treated Water混匀后微离心，接着可以在65℃孵育5分钟，随后立即置于冰浴以打开RNA的一些比较稳定的二级结构。RNaseInhibitor可以视其本身的情况加入适当的量，例如0.5μl或其他适当体积。在加入其他体积时，DEPC-treated Water的用量需适当调整。dNTP浓度不同时使用的体积需作适当调整，此时DEPC-treated Water的用量需适当调整。b.轻轻混匀(可以用Vortex在最低速度轻轻混匀或用移液器吹打混匀)，随后离心沉淀液体。c.如果使用Oligo(dT)18作为引物或使用基因特异性引物，在42℃孵育60分钟。如果使用randomhexamer(随机六聚体)作为引物，先在25℃孵育10分钟，随后在42℃孵育60分钟。注意：对于GC含量比较高的RNA模板的反转录反应，可以设置为45℃孵育60分钟。d.70℃孵育10分钟以失活RT M-MuLV反转录酶并终止反转录反应。说明：对于长片段的cDNA不推荐采用加热的方法失活RT M-MuLV反转录酶，这种操作可能会导致部分长片段DNA被剪切。e.反转录产物可以直接用于后续的PCR等反应，也可以-20℃冻存以备以后使用。用于后续PCR反应时，如果PCR的反应体系为50微升，则推荐使用2微升反转录产物。2.其他用途请自行参考M-MuLV反转录酶的相关文献资料进行。常见问题：1.总RNA反转录产物电泳观察不到。反转录产物由于是从模板反转录而获得，而模板的量本身比较低，反转录的量通常还要少于模板量，因此通常总RNA的反转录产物直接电泳观察是观察不到的。2.反转录产物通过PCR扩增没有特异性条带。PCR扩增没有获得特异性条带时建议先使用actin、GAPDH等作为内参进行PCR扩增，看是否可以成功扩增。如果可以成功，则说明PCR扩增体系没有问题，此时通常是目的基因的引物设计欠佳，当然也有可能是反转录产物质量欠佳。如果内参不能被很好地扩增，则有可能PCR体系存在问题或反转录产物质量欠佳。
EnzymoPure™ M-MuLV Reverse Transcriptase is a modified and optimized Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase. It is a DNA polymerase that uses RNA or DNA as template to synthesize complementary DNA strands in the presence of primers. It also has Ribonuclease H (RNase H) activity, which can specifically degrade the RNA in RNA-DNA hybrids, but not single-stranded RNA or double-stranded RNA.Features
Application：
First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling; RNA analysis by primer extension.
Source：
Recombinant protein expressed in E. coli. The RT M-MuLV reverse transcriptase is encoded by the mutation-optimized pol gene encoding M-MuLV reverse transcriptase.Enzyme Activity: One unit of the enzyme incorporates 1 nmol of dTMP into a polynucleotide fraction in 10 min at 37℃. Enzyme activity is assayed in 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 10 mM DTT, 40 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [3H]-dTTP, 0.4 mM polyA•oligo(dT)12-18.Purity: Free of DNA endonuclease, DNA exonuclease, phosphatase, and RNase other than the RNase H enzyme activity contained in the RTTM M-MuLV Reverse Transcriptase.
Enzyme storage buffer：
50 mM Tris, pH 8.3, 100mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% Triton X-100 and 50% glycerol.
Inactivation or inhibition：
M-MuLV Reverse Transcriptase can be inactivated by incubation at 70℃ for 10 minutes, or inhibited by chelating agents including EDTA and EGTA, inorganic phosphate, pyrophosphate, and polyamine.This product is sufficient for 10 reverse transcription reactions when used in a reaction volume of 20µl.
Precautions：
Please refer to the instructions for reverse transcription of RNAs with high GC content.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1.First-strand cDNA Synthesis:a. Set up the reaction in a nuclease-free PCR tube as follows:RNA Template (one of the three types of RNA)Total RNA0.1-5μgPoly(A) RNA/mRNA10-500ngSpecific RNA0.01pg-500ngPrimer (one of the three types of primers)Oligo(dT)180.5μg (or100pmol)random hexamer0.2μg (or100pmol)Gene specific primer15-25pmol(optional) For RNAs with high GC content or complex secondary structures, incubate the mixture of primer and template at 65℃ for 5 minutes, and immediately put it on ice to disrupt RNA secondary structures.DEPC-treated Water-To 13.7μl *Reaction Buffer (5X)-4μlRNase Inhibitor-0.5μl **dNTP Mix (25 mM each)-0.8µl ***RTTM M-MuLV Reverse Transcriptase-1µlTotal Volume-20µl* ‘To 13.7μl' means filling the mixture of template and primer to a total volume of 13.7μl with DEPC-treated Water. ** The volume of RNase Inhibitor may vary depending on the type of RNase Inhibitor used. If the volume of RNase Inhibitor is not 0.5μl, adjust the volume of DEPC-treated Water accordingly.*** The volume of dNTP mix varies depending on the concentration of dNTP stock. If the volume of dNTP is not 0.8µl, adjust the volume of DEPC-treated Water accordingly.b. Mix well by vortex or pipetting gently, centrifuge briefly to collect liquid at the bottom of PCR tube.c. If using Oligo(dT)18 or gene-specific primers, incubate the reaction mixture at 42℃ for 60 minutes. If random hexamer is used, incubate at 25℃ for 10 minutes followed by 60 minutes at 42℃. Note: For RNA templates with high GC content, incubate the reaction at 45℃ for 60 minutes.d. Incubate at 70℃ for 10 minutes to stop the reaction. Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. In such a case, phenol-chloroform extraction or column purification can be considered.e. The reverse transcription products can be used directly for subsequent experiments such as PCR, or stored at -20℃ for future use. We recommend using 2μl reverse transcription products in a PCR reaction volume of 50μl.For other uses, please refer to the relevant literature of M-MuLV reverse transcriptase.FAQ:1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomenon, because the amount of RNA template is low, and the amount of reverse transcription products in different size is even lower.2. No specific product can be amplified from the reverse transcription product.a. To exclude the problem of PCR reaction system or reverse transcription product, use gene-specific primers to amplify internal reference genes, such as actin and GAPDH. Reference genes can be amplified but not the target gene, indicating primers of target gene are not well designed or the expression of the target gene is too low to be detected. b. Inappropriate primer is used for reverse transcription. Random hexamer instead of Oligo(dT)18 should be used for the reverse transcription of bacterial total RNA which does not have poly(A) tails. Gene-specific primers used for reverse transcription must be well designed.
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