| Description | EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) 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 EnzymoPure™II M-MLV Reverse Transcriptase (RNase H-) produced by aladdin is an optimized Moloney Murine Leukemia Virus (M-MLV) 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. It is one of the most widely used reverse transcriptase.sApplication：First strand cDNA synthesis using total RNA or mRNA as template; DNA probe labeling with fluorescence, biotin, digoxin or isotope; RNA analysis by primer extension.This product is highly cost-effective. The RT II M-MLV reverse transcriptase (RNase H-) is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene fragment of M-MLV with RNase H coding region removed. The enzyme has been engineered to have better thermal stability and higher reverse transcription activity.Definition of enzyme activity: One unit of the enzyme incorporates 1nmol of dTTP into acid-precipitable material in 10 min at 37℃ using poly(A)•oligo(dT)12-18 as template-primer. The reaction system contains 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT, 0.5mM [3H]-dTTP and 0.4 mM poly(A)•oligo(dT)12-18.Purity: Free from DNA endonuclease, DNA exonuclease, phosphoesterase and RNase.Enzyme storage buffer: 20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition: RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV 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. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) (, #) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, ), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the S, M and L are sufficient for 50, 250 and 1000 reactions, respectively.Enzyme storage buffer：20mM Tris-HCl (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01%(v/v) NP-40 and 50% (v/v) glycerol.Inactivation or inhibition：RT II M-MLV Reverse Transcriptase (RNase H-) can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product has high reverse transcription activity, and is able to synthesize long cDNA. The RT II M-MLV 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. It can easily synthesize cDNA up to 8kb (Figure 1). The maximum length of synthesized cDNA by this product can exceed 10kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template that was synthesized by the RT II M-MLV reverse transcriptase (RNase H-). cDNA ranging from 0.2kb to 8kb in length can be synthesized high efficiently.This product exhibits high thermal stability. This product has an optimum working temperature of 42-45ºC, but it is still highly active at 50ºC (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA templates that were synthesized by the RT II M-MLV reverse transcriptase (RNase H-) and by the M-MLV reverse transcriptase (RNase H-) (Beytome, D7159), respectively. 500ng of total RNA extracted from NIH3T3 cells was reverse transcribed by reverse transcriptase in a reaction volume of 20µl at difference temperatures as indicated in the figure. One microliter of each reverse transcription reaction product was taken for PCR amplification.The concentration of this product is 200U/µl. When 20µl of reverse transcription reaction volume is used, the D7160S, D7160M and D7160L are sufficient for 50, 250 and 1000 reactions, respectively.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 synthesis.a. 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. Template (one of the three types of RNA)Total RNA0.1ng-5µgPoly(A) RNA/mRNA10pg-0.5µgSpecific RNA0.01pg-0.5µgPrimer (one of the three types of primer)Oligo(dT)18 Primer0.5µg (or 100pmol)Random Hexamer Primer0.2µg (or 100pmol)Gene-specific Primer15-25pmol(Optional) For RNAs with high GC content (e.g. ＞55%) 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 Water-To 13.7µl*Reaction Buffer (5X)-4µlRNase Inhibitor (40U/µl)-0.5µldNTP Mix (25mM each)-0.8µl**RT II M-MLV Reverse Transcriptase (RNase H-)-1µl***Total 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 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.*** If gene specific primers or Oligo(dT)18 primers are used to reverse transcribe the cDNA over 5kb, the volume of RT II M-MLV reverse transcriptase (RNase H-) should be increased to 2µl.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 60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, carry out incubation at 25℃ for 10min, followed by incubation at 42℃ for 60min. Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃ for 60min in order to improve the reverse transcription efficiency.d. Stop the reverse transcription by incubating the reaction at 80ºC for 10min to inactivate RT II M-MLV 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. If reference genes can be amplified but not the target gene, it indicates primers of target gene are not well designed, the expression of the target gene is too low to be detected, or the reverse transcription efficiency is low. b. Template RNA may be degraded. The integrity of total RNA can be checked by agarose gel or on-chip electrophoresis. Intact total RNA exhibits sharp, clear 28S and 18S rRNA bands, and the 28S rRNA band should be approximately twice as intense as the the 18S rRNA band. A ratio less than 2 indicates the degradation of total RNA and new total RNA should be prepared. c. RNA samples may contain some components that inhibit the activity of reverse transcriptase. Those contaminants include phenol, SDS, EDTA, guanidine salts, phosphoric acid, pyrophosphoric acid, polyamine, and spermidine etc, which can be removed effectively by column purification or precipitation, washing, and redissolution. Total RNA extracted by Zol (, #R0011) or Trizol (, #R0016) usually can meet the requirements of reverse transcription.d. Insufficient templates for reverse transcription. When DNase I used to remove the residual DNA in RNA sample is subjected to heat-inactivation prior to reverse transcription, EDTA should be added to RNA sample at a final concentration of 2.5mM to protect RNA from degradation under high temperature. Additionally, to amplify a specific gene, it is necessary to extract RNA from tissues in which the target gene is highly expressed. e. 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.f. For RNA template with high GC content or secondary structures, increase the reverse transcription temperature to 45-50℃ to improve the reverse transcription efficiency... 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 | Recombinant Human Serum Albumin (rHSA) is an active compound and possesses an identical conformation to plasma derived HSA. Recombinant Human Serum Albumin (rHSA) has no difference between rHSA and plasma derived HSA. Recombinant Human Serum Albumin (rHSAAppearance:SolidBiological Activity:Recombinant Human Serum Albumin (rHSA) is an active compound and possesses an identical conformation to plasma derived HSA. Recombinant Human Serum Albumin (rHSA) has no difference between rHSA and plasma derived HSA. Recombinant Human Serum Albumin (rHSAAppearance:SolidBiological Activity:Recombinant Human Serum Albumin (rHSA) is an active compound and possesses an identical conformation to plasma derived HSA. Recombinant Human Serum Albumin (rHSA) has no difference between rHSA and plasma derived HSA. Recombinant Human Serum Albumin (rHSA... Read More | Purity>97% by SDS-PAGE and HPLC analyses.FunctionPlays an important role in the organization of the cytoskeleton (By similarity). Binds to and sequesters actin monomers (G actin) and therefore inhibits actin polymerization. Seraspenide inhibits the entry of hematopoeitic pluripotent stem cells Purity>97% by SDS-PAGE and HPLC analyses.FunctionPlays an important role in the organization of the cytoskeleton (By similarity). Binds to and sequesters actin monomers (G actin) and therefore inhibits actin polymerization. Seraspenide inhibits the entry of hematopoeitic pluripotent stem cells into the S-phase... Read More | Inquire |