| Description | EnzymoPure™M-MuLV Reverse Transcriptase (RNase H-) is an optimized Moloney Murine Leukemia Virus (M-MuLV) 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 EnzymoPure™M-MuLV Reverse Transcriptase (RNase H-) is an optimized Moloney Murine Leukemia Virus (M-MuLV) 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. Different from common M-MuLV reverse transcriptase, the EnzymoPure™M-MuLV 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. EnzymoPure™M-MuLV reverse transcriptase (RNase H-) is one of the most widely used reverse transcriptase for synthesizing cDNA.FeaturesApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling; RNA analysis by primer extension; fluorescent probe labeling for DNA microarray analysis.Source：Recombinant protein expressed in E. coli. The RT M-MuLV reverse transcriptase (RNase H-) is encoded by the mutation-optimized pol gene encoding M-MuLV reverse transcriptase.Enzyme activity: One unit of the enzyme incorporates 1nmol of dTMP into a polynucleotide fraction in 10min at 37℃. Enzyme activity is assayed in 50mM Tris-HCl (pH8.3), 6mM MgCl2, 10mM DTT, 40mM KCl, 0.5mM dTTP, 0.4MBq/ml [3H]-dTTP, 0.4mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Storage buffer: 50mM Tris (pH8.3), 100mM NaCl, 1mM EDTA, 5mM DTT, 0.1% Triton X-100 and 50% glycerol.Reaction Buffer (5X): 250mM Tris (pH8.3 at 25℃), 250mM KCl, 20mM MgCl2, 50mM DTT.Inactivation or inhibition：RT M-MuLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 70℃ for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.The concentration of this product is 200U/µl. When using a reaction volume of 20µl, this product is sufficient for 10 reactions.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 synthesisa. 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. RNA Template (one of the three types of RNA)Total RNA0.01-5µgPoly(A) RNA/mRNA1-500ngSpecific RNA0.01pg-500ngPrimer (one of the three types of primers)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(optional) For RNAs with high GC content 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 WaterTo 13.7µl*Reaction Buffer (5X)4µlRNase Inhibitor (40U/µl)0.5µl**dNTP Mix (25 mM each)0.8µl***RT M-MuLV Reverse 1µlTotal Volume20µl* ‘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 less than 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 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 10-60min if Oligo(dT)18 or gene-specific primer is used. If random hexamer is used, carry out incubation at 25ºC for 10min, followed by incubation at 42℃ for 60 min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 45℃ for 60min.d. Stop the reverse transcription by incubating the reaction at 70℃ for 10min to inactivate the RT M-MuLV 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. 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 | Mammalian lactate dehydrogenases (LDH) exist as five tetrameric isozymes composed of combinations of two different subunits. The H subunit predominates in heart muscle, which is geared for aerobic oxidation of pyruvate. The M subunit predominates in skeletal muscle and is concerned more with Mammalian lactate dehydrogenases (LDH) exist as five tetrameric isozymes composed of combinations of two different subunits. The H subunit predominates in heart muscle, which is geared for aerobic oxidation of pyruvate. The M subunit predominates in skeletal muscle and is concerned more with anaerobic metabolism and pyruvate reduction.Catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+Recombinant rabbit muscle Lactate Dehydrogenase produced in E.Coli. Chromatographically purified. A lyophilized powder... Read More | Inorganic pyrophosphates are inevitably produced in the process of mRNA transcription in vitro. These substances have a great inhibitory effect on transcription. Inorganic pyrophosphatase (PPase) can hydrolyze the inorganic pyrophosphates produced in nucleic acid amplification experiments, promote Inorganic pyrophosphates are inevitably produced in the process of mRNA transcription in vitro. These substances have a great inhibitory effect on transcription. Inorganic pyrophosphatase (PPase) can hydrolyze the inorganic pyrophosphates produced in nucleic acid amplification experiments, promote the shift of reaction equilibrium to the product generation end, and increase the amount of products.The molecular weight of PPase (pyrophosphatase, inorganic, inorganic pyrophosphatase) is about 63kd, which can catalyze the hydrolysis of inorganic pyrophosphate to produce orthophosphate: P2O74_+H2O+PPase→2HPO42_. In the nucleic acid amplification experiment, PPase can hydrolyze the inorganic pyrophosphate generated with the reaction to avoid its inhibition on the reaction system. The removal of pyrophosphate can shift the reaction equilibrium to the product generation end.This product is a GMP level recombinant inorganic pyrophosphatase (yeast source) expressed by large-scale fermentation of E. coli. It is produced with raw and auxiliary materials of medicinal specifications, and the host protein residue and nucleic acid residue are strictly controlled. The product production and quality management procedures in line with GMP specifications ensure that the production process and all raw and auxiliary materials can be traced.Quality requirements project standard appearance Clear liquid Visible foreign matter Compliance with regulations PH value 7.5±8.5 activity 98U/ml-102U/ml purity ≥95% Endonuclease residues Degradation of substrate shall not exceed 10% Exonuclease residues Degradation of substrate shall not exceed 10% RNase residue Degradation of substrate shall not exceed 10% Bacterial endotoxin content ≤10EU/ml Exogenous DNA residue ≤100pg/mg Host protein residue ≤50ppm Mycoplasma detection negative Heavy metal residues ≤10ppm Follow the following specifications1. ISO 9001:2015, certified facility。2. GMP appendix - cell therapy products State Drug Administration.3. general introduction to human gene therapy - Chinese Pharmacopoeia 2020, National Pharmacopoeia Committee.4. USP chapter <1043>, adjuvant materials for cell, gene, and tissue engineered products.5. USP chapter <92>, growth factors and cytokines used in cell therapy manufacturing.6. Ph. Eur. General chapter 5.2.12, raw materials of biological origin for the production of cell-based and gene therapy medical products.Product features1. hydrolyze inorganic pyrophosphate.2. DNA synthesis: significantly enhance DNA replication ability.3. RNA synthesis: increase RNA production in in vitro transcription reaction.4. The optimal reaction temperature is 25℃, and the enzyme can be inactivated at 65℃ for 10min.Product usage1. optimize RNA transcription: improve the RNA yield of in vitro transcription reaction.2. remove PPI contamination from reagents for SNP genotyping by pyrophosphate assay.3. promote the synthesis of protein, RNA and DNA.4. catalyze the reaction of PPI + H2O → 2pi.5. ssr-pcr optimization：Improve efficiency and increase DNA production.Activity definitionCatalytic inorganic pyrophosphate formation 1 per minute under standard reaction conditions µ The amount of enzyme required for mol phosphate was defined as 1 active unit.Preservation system20 mM Tris-HCl; 100 mM NaCl; 1 mM DTT; 0.1 mM EDTA; 50% (v/v) Glycerol; pH 8.0。 Storage temperature-20±5 ℃。Matters needing attention1. the enzyme has activity in various reaction buffers. Generally, the enzyme can be directly added in HDA, lamp and other experiments.2. the dosage of the enzyme needs to be optimized in different experiments, usually adjusted at the concentration of 0.05~1u/ml.3. the optimum reaction temperature of the enzyme was 25 ℃, and it was active at 16~37 ℃, and the enzyme could be inactivated at 65 ℃ for 10min.4. cofactor: mg2+ is necessary for enzyme activity... Read More | Purity: >95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： CD4, also known as L3T4, T4, and W3/25, is an approximately 55 kDa type I transmembrane glycoprotein that is expressed predominantly on thymocytes and a subset of mature T lymphocytes. It is a standard Purity: >95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： CD4, also known as L3T4, T4, and W3/25, is an approximately 55 kDa type I transmembrane glycoprotein that is expressed predominantly on thymocytes and a subset of mature T lymphocytes. It is a standard phenotype marker for the identification of T cell populations. Mature human CD4 consists of a 371 amino acid (aa) extracellular region containing four immunoglobulin-like domains, a 22 aa transmembrane segment, and a 40 aa cytoplasmic domain. Within the ECD, human CD4 shares approximately 52% aa sequence identity with mouse and rat CD4. CD4 is expressed along with CD8 on double positive T cells during their development in the thymus. Either CD4 or CD8 expression is then lost, giving rise to single positive (SP) CD4+ or CD8+ mature T cells. CD4+ SP cells, also known as T helper cells, further differentiate into multiple subsets of CD4+ cells including Th1, Th2, Th17, Tfh, and Treg cells which regulate humoral and cellular immunity. CD4 is reexpressed on circulating CD8+ T cells upon activation and contributes to their cytotoxic effector activity. In human, CD4 is additionally expressed on macrophages, neutrophils, monocytes, NK cells, and neurons and glial cells in the brain. Similar CD4 distribution between species cannot be assumed as demonstrated by its presence on macrophages in human and rat but not in mouse. CD4 binds directly to MHC class II molecules on antigen presenting cells. This interaction contributes to the formation of the immunological synapse which is focused around the TCR-MHC class II-antigenic peptide interaction. Palmitoylation of two cysteine residues in the cytoplasmic tail of CD4 promotes the localization of CD4 in lipid rafts and its ability to augment TCR signaling via activation of the tyrosine kinase Lck. CD4 also functions as a chemotactic receptor for IL-16 and, in human, as a co-receptor for the gp120 surface glycoprotein of HIV-1... Read More | Purity>95% SDS-PAGE.FunctionProbable cell adhesion protein |