| Description | 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) 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.FeaturesApplication：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... Read More | Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high amplification efficiency and low mismatch rate, and can efficiently amplify DNA fragments. Most of the PCR products amplified with this product contain an "A" base at the 3 'end, which can be directly used for T/A cloning. This product is suitable for conventional PCR reactions and gene cloning reactions that require high fidelity. E665597Component500 UStorageE665597AEs Taq DNA Polymerase, 5 U/µL 100 µL -20℃. Avoid freeze/thaw cycle.E665597B10×PCR Buffer 1.8 mL -20℃. Avoid freeze/thaw cycle.Activity definition:Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.Quality control:After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes in activity.1. PCR reaction system Reagent 50 µlReaction system Final concentration 10×PCR Buffer 5 µL 1× dNTP Mix，10 mM each 1 µL 200 µM each Forward Primer，10 µM 2 µL 0.4 µM Reverse Primer，10 µM 2 µl 0.4 µM Template DNA <0.5 µg <0.5 µg/50 µl Es Taq DNA Polymerase，5 U/µl 0.25-0.5 µl 1.25-2.5U/50 µl ddH2O up to 50 µL /Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions Step Temperature Time / Pre denaturation 94℃ 2 min / Denaturation 94℃ 30 s 25-35 cycles Anneal 55-65℃ 30 s 25-35 cycles Extend 72℃ 30 s 25-35 cycles Finally extended 72℃ 2 min / Attention:1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Es Taq DNA Polymerase in this product is 2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible... Read More | Inquire | Purity> 97 % by SDS-PAGE and HPLC analyses.Additional sequence informationThis product is for the mature full length protein. The signal peptide is not included.FunctionInhibits hemopoiesis and stimulates chemotaxis. Chemotactic in vitro for thymocytes and activated T-cells, but not for B-cells, Purity> 97 % by SDS-PAGE and HPLC analyses.Additional sequence informationThis product is for the mature full length protein. The signal peptide is not included.FunctionInhibits hemopoiesis and stimulates chemotaxis. Chemotactic in vitro for thymocytes and activated T-cells, but not for B-cells, macrophages, or neutrophils. Shows preferential activity towards naive T-cells. May play a role in mediating homing of lymphocytes to secondary lymphoid organs... Read More | Purity: >90%, by SDS-PAGE visualized with Coomassie® Blue Staining. Function：Actin cross-linking/gelling protein (By similarity). Involved in calcium interactions and contractile properties of the cell that may contribute to replicative senescence |