| 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 | Inquire | Product DescriptionEndo F1 cleaves Asparagine-linked high mannose and some hybrid oligosaccharides. Core fucosylation reduces the activity by 50 fold. Endoglycosidase F1 will hydrolyze sulfate containing high-mannose chains. It cleaves between the two N-acetylglucosamine residues in the Product DescriptionEndo F1 cleaves Asparagine-linked high mannose and some hybrid oligosaccharides. Core fucosylation reduces the activity by 50 fold. Endoglycosidase F1 will hydrolyze sulfate containing high-mannose chains. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.Molecular weight 32,000 daltonsContents60 µl aliquot of enzyme (1 U) in 20 mM Tris-HCl, pH 7.5Included with 20 µL and 60 µL pack sizes:5x Reaction Buffer – 250 mM sodium phosphate, pH 5.5Specific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured Ribonuclease B (RNase B) in 1 minute at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).FormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, pH 7.5StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. SpecificityEndo F1 cleaves Asparagine-linked high mannose or hybrid oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Quality & PurityEndo F1 is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Directions for use1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 38 µl final volume with de-ionized water.2. Add 10 µl 5x Reaction Buffer 5.53. Add 2.0 µl of Endo F1 to the reaction. Incubate 1 hour or more at 37°C.Monitor cleavage by SDS-PAGE... Read More | GoldStar Probe Mixture is a premixed system specifically designed for real-time fluorescence quantitative PCR using probe methods (TaqMan, Molecular Beacon, etc.), with a concentration of 2 x, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, and Mg2+. The operation is simple and convenientGoldStar Probe Mixture is a premixed system specifically designed for real-time fluorescence quantitative PCR using probe methods (TaqMan, Molecular Beacon, etc.), with a concentration of 2 x, containing GoldStar Taq DNA Polymerase, PCR Buffer, dNTPs, and Mg2+. The operation is simple and convenient. Mainly used for detecting genomic DNA target sequences and RNA reverse transcription cDNA target sequences, such as gene expression analysis, copy number analysis, SNP genotype analysis, etc., suitable for fluorescence quantification using different types of probe methods. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, novel and highly efficient hot start enzyme. It has no polymerase activity at room temperature, effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. The enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique combination of PCR buffer system and hot start enzyme significantly improves the amplification efficiency of PCR, with stronger fluorescence signal and higher sensitivity, which can detect single copy templates. By using this product, a wider linear range can be obtained, resulting in more accurate quantification of the target gene. Suitable for all fluorescence quantitative PCR instruments that do not require ROX as a calibration dye.ROX dye is used to correct the fluorescence signal error between wells in quantitative PCR instruments, and is generally used in Real Time PCR amplification instruments from companies such as ABI and Stratagene. The excitation optical systems of different instruments vary, so the concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (CW0932): Roche LightCycle 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96, etc.Instrument requiring Low ROX calibration (CW2625): 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, etc.Instruments that require High ROX calibration (CW2626): ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.G665832Component5 mLStorageG665832A2×GoldStar Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.G665832BddH2O5×1 mL -20℃. Avoid freeze/thaw cycle. Notes:1. Before use, please gently mix upside down to avoid foaming, and use after briefly centrifugation.2. Avoid repeated freezing and thawing of this product, as repeated freezing and thawing may cause a decrease in product performance. This product can be stored for a long time at -20 ℃, away from light. If frequent use is required in the short term, it can be stored at 2-8 ℃.Usage:The following are examples of conventional PCR reaction systems and reaction conditions. In practical operation, corresponding improvements and optimizations should be made based on different templates, primer structures, and target fragment sizes.1. PCR reaction system Reagent 50 µl Reaction system Final concentration 2×GoldStar Probe Mixture 25 µl 1 × Forward Primer，10 µM 1 µl 0.2 µM¹⁾ Reverse Primer，10 µM 1 µl 0.2 µM¹⁾ Probe，10 µM 1 µl 0.2 µM²⁾ Template DNA 2 µl³⁾ / 50×Low ROX or High ROX（optional）⁴⁾ 1 µl 1 × ddH2O up to 50 µl / Attention:1) Typically, the primer concentration is 0.2 µ M can achieve good results, ranging from 0.1 to 1.0 µ M serves as a reference for setting the range.2) The 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. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.3) The amount of DNA template is usually based on 10-100 ng genomic DNA or 1-10 ng cDNA as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the template to determine the optimal template usage.4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.2. PCR reaction programAttention! The pre denaturation reaction of this product must be completed at 95 ℃ for 10 minutes!Two step PCR Step Temperature Time / Pre denaturation 95℃ 10 min¹⁾ / Denaturation 95℃ 15 s 35-40 cycles Annealing/Extension ²⁾ 60℃ 1 min 35-40 cycles Attention:1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 10 minutes.2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference... Read More | Seals and prevents freezing of stopcocks and ground-glass joints in high-vacuum systems at pressures less than 10-6 mm Hg. Heat stable (?40 to 260 °C), low vapor pressure, and chemically resistant. Colorless. 5.3 oz. tube |