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 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