| Description | EnzymoPure™III M-MLV reverse transcriptase is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize the first-strand complementary DNA strands (cDNA) rapidly and efficiently in the presence of primers. It is optimized to have a high thermal stability and high fidelityEnzymoPure™III M-MLV reverse transcriptase is a DNA polymerase that uses single stranded RNA or DNA as template to synthesize the first-strand complementary DNA strands (cDNA) rapidly and efficiently in the presence of primers. It is optimized to have a high thermal stability and high fidelity, and can synthesize long cDNA up to 12kb. Additionally, this product has RNase H activity that selectively degrades the RNA strand of an RNA-DNA hybrid, thus facilitating the synthesis of second-strand cDNA subsequently.EnzymoPure™III M-MLV Reverse Transcriptase is one of the most widely used high-quality reverse transcriptase. It is mainly used for synthesizing first-strand cDNA with total RNA or mRNA template. The obtained cDNA can be used directly for PCR, real-time PCR, second-strand cDNA synthesis and construction of cDNA libraries, etc. Moreover, it can also be used to perform RNA analysis by primer extension, or label DNA probes with fluorescence, biotin, digoxin or isotope.EnzymoPure™III M-MLV Reverse Transcriptase is a high efficient reverse transcriptase, requiring 10 minutes only to synthesize cDNA no longer than 3kb. To synthesize cDNA longer than 3kb, especially longer than 6kb, 60min of reverse transcription is recommended.EnzymoPure™III M-MLV Reverse Transcriptase is a high efficient reverse transcriptase, requiring 10 minutes only to synthesize cDNA no longer than 3kb. To synthesize cDNA longer than 3kb, especially longer than 6kb, 60min of reverse transcription is recommended.This product is highly cost-effective. EnzymoPure™III M-MLV Reverse Transcriptase is a recombinant reverse transcriptase expressed and purified from E. coli transformed with the expression plasmids carrying the pol gene of M-MLV with RNase H coding region included. The enzyme has been engineered to have better thermal stability and higher reverse transcription efficiency, and produce longer reverse transcript.Definition of enzyme activityInactivation or inhibition：RT III M-MLV Reverse Transcriptase can be inactivated by incubation at 80ºC for 10 minutes, or inhibited by EDTA, EGTA, inorganic phosphates, pyrophosphates and polyamine.This product is able to synthesize long cDNA. It enables easy reverse transcription of genes less than 8kb (Figure 1). The maximum length of cDNA can be up to 12kb. Figure 1. Agarose gel electrophoresis of PCR products amplified from the cDNA template synthesized with the RT III M-MLV Reverse Transcriptase (, #D7176). cDNA ranging from 0.2kb to 12kb in length can be synthesized high efficiently.RT III M-MLV Reverse Transcriptase has good thermal stability and high reverse transcription efficiency. It has an optimum working temperature of 42℃, but it is still highly active at 50℃. Reverse transcription at higher temperature can significantly improve the reverse transcription of RNA with high GC content and complex secondary structures. This reverse transcriptase has a high reverse transcription rate, requiring 30 minutes only to complete the synthesis of cDNA less than 6kb, and 10min only to obtain cDNA less than 3kb (Figure 2). Figure 2. Agarose gel electrophoresis of PCR products amplified from the cDNA template synthesized by the RT III M-MLV Reverse Transcriptase (, #D7176). 2µg total RNA extracted from HEK293T cells was reverse transcribed in a reaction volume of 20µl at different temperatures for different duration of reverse transcription, as indicated in the figure. After reverse transcription, 1µl reverse transcription product was used to amplify YWHAZ and ADAR1 genes of 2.6kb and 6.0kb, respectively.The concentration of RT III M-MLV Reverse Transcriptase is 200U/µl. When 20µl of reverse transcription volume is used, the D7176S, D7176M and D7176L are sufficient for 50, 250 and 1000 reactions, respectively.Precautions：Please refer to the instructions for reverse transcription of RNA 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 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 Primer15-25pmolDEPC-treated Water-To 13.7µl*(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.Reaction Buffer (5X)-4µlRNase Inhibitor (40U/µl)-0.5µldNTP Mix (25mM each)-0.8µl**RT III M-MLV Reverse Transcriptase-1µlTotal Volume-To 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 with 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 42℃for 10-60min if Oligo(dT)18 primer or gene-specific primer is used. If random hexamer is used, incubate at 25℃for 10min, followed by incubation at 42℃for 10-60min. For cDNA less than 3kb, 10-minute incubation is usually sufficient. For cDNA between 3kb and 6kb, we recommend 30 minutes incubation. While for cDNA exceeding 6kb in length, 60-minute incubation is recommended. When using random hexamers for the reverse transcription which will be followed by qPCR experiments, 10-minute incubation is sufficient regardless of the length of cDNA.Note: For RNA template with high GC content or secondary structures, incubate the reaction at 50℃for 60min. The RT III M-MLV Reverse Transcriptase maintains good reverse transcriptase activity at 50ºC, and reverse transcription at a higher temperature effectively reduce the interference of secondary structures.d. Stop the reverse transcription by incubating the reaction at 80℃for 10min to inactivate RT III M-MLV Reverse Transcriptase. Note: Heat-inactivation of reverse transcriptase is not recommended for long cDNA over 5kb, as this method may cause shearing of long cDNA fragments. 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 0.8µl and 2µl reverse transcription products in a PCR reaction volume of 20µl and 50µl, respectively.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 | Inquire | Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high Es Taq DNA Polymerase is an optimized mixed enzyme of Taq and Pfu DNA Polymerase, with 5 '→ 3' DNA polymerase activity, 5 '→ 3' exonuclease activity, and 3 '→ 5' exonuclease activity. Compared with Taq DNA Polymerase, Es Taq DNA Polymerase has excellent performance of high amplification efficiency and low mismatch rate, and can efficiently amplify DNA fragments. Most of the PCR products amplified with this product contain an "A" base at the 3 'end, which can be directly used for T/A cloning. This product is suitable for conventional PCR reactions and gene cloning reactions that require high fidelity. E665597Component500 UStorageE665597AEs Taq DNA Polymerase, 5 U/µL 100 µL -20℃. Avoid freeze/thaw cycle.E665597B10×PCR Buffer 1.8 mL -20℃. Avoid freeze/thaw cycle.Activity definition:Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.Quality control:After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes in activity.1. PCR reaction system Reagent 50 µlReaction system Final concentration 10×PCR Buffer 5 µL 1× dNTP Mix，10 mM each 1 µL 200 µM each Forward Primer，10 µM 2 µL 0.4 µM Reverse Primer，10 µM 2 µl 0.4 µM Template DNA <0.5 µg <0.5 µg/50 µl Es Taq DNA Polymerase，5 U/µl 0.25-0.5 µl 1.25-2.5U/50 µl ddH2O up to 50 µL /Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions Step Temperature Time / Pre denaturation 94℃ 2 min / Denaturation 94℃ 30 s 25-35 cycles Anneal 55-65℃ 30 s 25-35 cycles Extend 72℃ 30 s 25-35 cycles Finally extended 72℃ 2 min / Attention:1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Es Taq DNA Polymerase in this product is 2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible... 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%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description:Cyclophilin B (SCYLP, CyPB, and peptidyl-prolyl cis-trans isomerase B) is a 24 kDa glycoprotein member of the B subfamily of the cyclophilin-type PPIase family of molecules. It is both secreted and retained in Purity: >95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description:Cyclophilin B (SCYLP, CyPB, and peptidyl-prolyl cis-trans isomerase B) is a 24 kDa glycoprotein member of the B subfamily of the cyclophilin-type PPIase family of molecules. It is both secreted and retained in the ER. When secreted, it mediates chemotaxis and T cell adhesion to fibronectin. This is likely due to its prolyl cis/trans isomerase activity. Intracellularly, Cyclophilin B appears to serve as a molecular chaperone for molecules destined for secretion. It does so via stabilization and facilitating the activity of additional chaperones. The human CyPB precursor is 216 amino acids (aa) in length. It contains a 25 aa signal sequence plus a 191 aa mature region. There is a partial heparin-binding sequence (aa 27‑34), a PPIase domain (aa 47‑204), and a C-terminal ER retention motif (aa 213‑216). Over aa 34‑216, the human and mouse sequences are 95% aa identical... 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