| Description | EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase without ribonuclease H (RNase H) activity. It is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize complementary DNA strands in the presence of primers. It is one of the most widely used reverse transcriptase.sApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling with fluorescence, biotin, digoxin or isotope; RNA analysis by primer extension.This product is highly cost-effective. The RT II M-MLV reverse transcriptase (RNase H-) is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene fragment of M-MLV with RNase H coding region removed. The enzyme has been engineered to have better thermal stability and higher reverse transcription activity.Definition of enzyme activity: One unit of the enzyme incorporates 1nmol of dTTP into acid-precipitable material in 10 min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. The reaction system contains 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP and 0.4 mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Enzyme storage buffer: 20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition: RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV Reverse Transcriptase (RNase H-) lacks RNase H activity, and does not degrade the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of long cDNA. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, ), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the S, M and L are sufficient for 50, 250 and 1000 reactions, respectively.Enzyme storage buffer：20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition：RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV Reverse Transcriptase (RNase H-) lacks RNase H activity, and does not degrade the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of long cDNA. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, D7159), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the D7160S, D7160M and D7160L are sufficient for 50, 250 and 1000 reactions, respectively.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 lab coat and disposable gloves during the operation.Instructions for Use：1. First-strand cDNA synthesis.a. Set up the first-strand cDNA synthesis reaction in a nuclease-free PCR tube on ice or at room temperature as follows. RNase Inhibitor and dNTP mix can be purchased from. Template (one of the three types of RNA)Total RNA0.1ng-5µgPoly(A) RNA/mRNA10pg-0.5µgSpecific RNA0.01pg-0.5µgPrimer (one of the three types of primer)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(Optional) For RNAs with high GC content (e.g. ＞55%) or complex secondary structures, incubate the mixture of primer and template at 65ºC 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 (40U/µl)-0.5µldNTP Mix (25mM each)-0.8µl**RT II M-MLV Reverse Transcriptase (RNase H-)-1µl***Total 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 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.*** If gene specific primers or Oligo(dT)18 primers are used to reverse transcribe the cDNA over 5kb, the volume of RT II M-MLV reverse transcriptase (RNase H-) should be increased to 2µl.b. Mix the reaction by vortex or pipetting gently, centrifuge briefly to allow liquid to accumulate at the bottom of PCR tube.c. Incubate the reaction at 42ºC for 60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, carry out incubation at 25℃ for 10min, followed by incubation at 42℃ for 60min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃ for 60min in order to improve the reverse transcription efficiency.d. Stop the reverse transcription by incubating the reaction at 80ºC for 10min to inactivate RT II M-MLV reverse transcriptase (RNase H-). 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.2. For other applications such as primer extension and probe labeling, please refer to reference related to M-MuLV reverse transcriptase (RNase H-).FAQ：1. The reverse transcription product of total RNA is invisible after electrophoresis.It is a normal phenomena, 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. If reference genes can be amplified but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, and spermidine etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol (, #R0011) or Trizol (, #R0016) usually can meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample is subjected to heat-inactivation prior to reverse transcription, EDTA should be added to RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. 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.f. For RNA template with high GC content or secondary structures, increase the reverse transcription temperature to 45-50℃ to improve the reverse transcription efficiency... Read More | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： This protein is a cell adhesion molecule involved in neuron-neuron adhesion, neurite fasciculation, outgrowth of neurites, etc | Purity>95% SDS-PAGE.FunctionB Cell Activating Factor Receptor (BAFF-R), also named tumor necrosis factor receptor superfamily member 13C, is a member of the TNFR superfamily. It is highly expressed in spleen, lymph node, and resting B cells and to some extent in activated B cells, resting CD4+ Purity>95% SDS-PAGE.FunctionB Cell Activating Factor Receptor (BAFF-R), also named tumor necrosis factor receptor superfamily member 13C, is a member of the TNFR superfamily. It is highly expressed in spleen, lymph node, and resting B cells and to some extent in activated B cells, resting CD4+ cells and peripheral blood leukocytes. BAFF receptor is a type III transmembrane protein containing a single extracellular phenylalanine-rich domain and binds with high specificity to BAFF (TNFSF13B). It enhances B-cell survival in vitro and is a regulator of the peripheral B-cell population. BAFF receptor/BAFF signaling plays a critical role in B cell survival and maturation... Read More | Purity>95% SDS-PAGEFunctionLipid transport protein in adipocytes. Binds both long chain fatty acids and retinoic acid. Delivers long-chain fatty acids and retinoic acid to their cognate receptors in the nucleus |