| 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 | Product Content:F665667Component5 mL40 mLStorageF665667A2×Flash PCR MasterMix (Dye) 5×1 mL 40×1 mL-20℃. Avoid freeze/thaw cycle.F665667BddH2O 5×1 mL40×1 mL-20℃. Avoid freeze/thaw cycle. Products Introduction This product is a premixed system consisting of a new Product Content:F665667Component5 mL40 mLStorageF665667A2×Flash PCR MasterMix (Dye) 5×1 mL 40×1 mL-20℃. Avoid freeze/thaw cycle.F665667BddH2O 5×1 mL40×1 mL-20℃. Avoid freeze/thaw cycle. Products Introduction This product is a premixed system consisting of a new high efficient fast DNA Polymerase, Mg2+, dNTPs, and PCR stabilizers and enhancers at 2× concentration. It is a new rapid DNA polymerase developed by CombiSigma with high amplification speed and stability. The extension speed is up to 5 s/kb, and the PCR can be completed in as little as 15 minutes, while longer fragments (>3 kb) or complex templates can be extended at a speed of 10-30 s/kb or a higher number of cycles. The unique MasterMix formula makes the whole reaction system very stable, while complex templates can be amplified effectively, and more than 98% of PCR amplification can be successful in one run. Simply add the DNA template and primers and top up with water to minimize human error, contamination and time.The dye (blue) has been added to the product and it is ready for electrophoretic detection at the end of the reaction. The PCR product is amplified with an 'A' base at the 3′ end and can therefore be used directly for T/A cloning and is suitable for use in the CombiVerge Seamless Cloning Kit, T4 Ligation Kit and sensory products.This product is mainly suitable for ultra-fast PCR, complex templates, complex secondary structures, gene cloning and large-scale genetic testing that requires high fidelity. quality control No exogenous nuclease activity was detected; no host residual DNA was detected by PCR; single-copy genes in various genomes could be amplified efficiently. UsageThe following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template, which should be improved and optimized according to the template, primer structure and size of the target fragment in actual operation.PCR reaction system Note: Please use the final concentration of 0.1-1.0 µM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the primer concentration can be increased; if a non-specific reaction occurs, the primer concentration can be decreased to optimize the reaction system.PCR reaction conditions Note: 1) Note: For simple templates, the pre-denaturation time can be controlled at 30 s-1 min, for complex templates such as bacterial fluids, the pre-denaturation time can be increased to 2 min.Optimization of parameter settings 1. Template DNA amount setting:Excessive amounts of template may result in non-specific amplification or smear. The recommended amount of template DNA in a 50 µl PCR reaction system is as follows:-Human genomic DNA 5 ng-500 ng-Escherichia coli genomic DNA 50 pg-100 ng-plasmid DNA 10 pg-1 ng 1. 30-35 number of cycles2. Primer concentration setting: The primer concentration can be set between 0.1 µM and 1.0 µM. A low primer concentration may result in low amplification products. Too high a primer concentration will inhibit specific amplification and may result in non-specific amplification.3. Annealing temperature setting: In general, the annealing temperature is 5℃ lower than the melting temperature of amplification primer Tm, so the annealing temperature can be lowered appropriately when the desired amplification efficiency cannot be obtained; the annealing temperature can be raised appropriately when non-specific reaction occurs. For complex templates, it is necessary to adjust the annealing temperature to achieve efficient amplification.4. Extension time setting: The extension time should be set according to the size of the amplified fragments. The following extension times are recommended: simple templates such as plasmids: 5-15 s/kb; regular genomes, cDNA templates: 10-15 s/kb; complex templates, crude templates: 20-30 s/kb; (the extension time should not be too short and should be at least 5 s/kb, but should not exceed 30 s/kb).5. Number of cycles: The number of cycles can be set according to the downstream application of the amplified product. If the number of cycles is too low, the amount of amplification will be insufficient; if the number of cycles is too high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield... Read More | Inquire | This product is a mixture of fast reverse transcription reagents. The 5 x EasyQuick RT MasterMix contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including EasyQuick RT Reversase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP, EQ-RT BufferThis product is a mixture of fast reverse transcription reagents. The 5 x EasyQuick RT MasterMix contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including EasyQuick RT Reversase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP, EQ-RT Buffer, etc. The reverse transcription efficiency of this product is high, and it can perform a good reverse transcription reaction on a small amount of RNA templates. The fluorescence quantitative template cDNA first strand synthesis can be completed in 15 minutes. This reagent kit is very convenient and fast to operate, and only RNA templates and water need to be added for reverse transcription reaction, making it particularly suitable for high-throughput detection.E665905Component200 TStorageE665905A5×EasyQuick RT MasterMix 400 µL-20℃. Avoid freeze/thaw cycle.E665905BRNase-Free Water 2×1 mL-20℃. Avoid freeze/thaw cycle. Product features1. Convenience: The ready to use reverse transcription Mix only requires the addition of RNA templates and water to initiate the reaction.2. Fast: Complete cDNA first strand synthesis in 15 minutes.3. High reverse transcription efficiency: The reverse transcription efficiency is above 90%.4. High sensitivity: PG level templates can also obtain high-quality cDNA.5. Read through complex templates: templates with high GC content and complex secondary structures.Matters needing attention1. During the operation, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended that operators wear masks and disposable gloves, frequently change gloves, and use specialized instruments and consumables.2. The reverse transcription system is prepared on ice for operation to prevent RNA degradation. The MasterMix of the reagent kit should be stored at -20 ℃ as soon as possible after use, and repeated freeze-thaw should be avoided as much as possible.3.10 µ The reaction system can be used up to 1 µ G Total RNA, if the amount of template RNA is greater than 1 µ g. Please expand the reaction system proportionally.4. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65 ℃ for 5 minutes on ice before proceeding with the next step, followed by brief centrifugation.Operation steps1. Thaw the template RNA on ice; After thawing the components of the reagent kit at room temperature, immediately place them on ice. Before use, vortex shake and mix each solution, and centrifuge briefly before use.2. Prepare the reaction system according to the following table (please prepare the reaction solution on ice), vortex shake and mix well, briefly centrifuge, and collect the solution on the tube wall to the bottom of the tube. Reagent 10 µl Reaction system Final concentration RNA Template X µl 1 pg～0.5 µg ¹⁾ 5×EasyQuick RT MasterMix ²⁾ 2 µl 1× RNase-Free Water up to 10 µl /Attention:1) If the total RNA content is greater than 1 µ g, please expand the reaction system proportionally.2) 5 x EasyQuick RT MasterMix contains Oligo (dT), Random Prime, RNase Inhibitor, dNTP Mixture, EQ-RT Buffer, etc. 3. Incubate at 37 ℃ for 15 minutes. 4. Incubate at 85 ℃ for 5 seconds to inactivate reverse transcriptase.5. After a brief centrifugation, place it on ice for subsequent experiments. If it needs to be stored for a long time, please place it at -20 ℃... Read More | Telomerase-IN-3 is a telomerase inhibitor, which directly targets hTERT promoter activity. hTERT is the key component for maintenance of telomerase activity.Form:Solid |