| 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 contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× Product contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× GoldStar Probe Mixture (UNG) is a premixed system dedicated to real-time fluorescence quantitative PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, containing GoldStar Taq DNA polymerase, PCR Buffer, dNTPs (dTTP is all replaced by dUTP), UNG enzyme and Mg2+, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequences and cDNA target sequences after RNA reverse transcription, such as gene expression analysis, copy number analysis and SNP genotype analysis. This product utilizes the dUTP-UNG anti-pollution system, which adds dUTP during the preparation of the PCR reaction system, thus forming an amplification product containing dU bases. This product can be eliminated by the UNG enzyme in the PCR system before the next PCR reaction. This effectively removes residual contamination of the PCR product and greatly reduces false positives due to contamination of the amplification product.UNG enzyme can be inactivated at the pre-denaturation step in the PCR cycle, and therefore will not affect the formation of new PCR products containing dU bases. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, new high-efficiency hot-start enzyme, which has no polymerase activity at room temperature, effectively avoiding non-specific amplification due to non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme must be incubated at 95°C for 10 minutes. The unique combination of PCR buffer system and hot-start enzyme significantly improves the amplification efficiency of PCR with stronger fluorescent signal and higher sensitivity to detect single-copy templates. A wider linear range and more accurate quantification of the target gene can be obtained by using this product.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 (G670150):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments requiring Low ROX calibration(G665780):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 requiring High ROX calibration(G665787):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attentionBefore use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.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 systemReagents50 µl Reaction systemfinal concentration2×GoldStar Probe Mixture（UNG）25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for 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 programCaution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!Two-step PCR:Note: 1) The hot-start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 10min. 2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out three-step PCR amplification.Three-step PCR:... Read More | Inquire | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More |