New England Biolabs, M0368X, ProtoScript® II Reverse Transcriptase

Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications Helicase-dependent Amplification,, cDNA Synthesis,, Reverse Transcription (cDNA Synthesis), Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 1 nmol of dTTP into acid-insoluble material in a total reaction volume of 50 μl in 10 minutes at 37°C using poly(rA)•oligo(dT) 18 as template. Reaction Conditions 1X ProtoScript® II Reverse Transcriptase Reaction Buffer Incubate at 42°C 1X ProtoScript® II Reverse Transcriptase Reaction Buffer 50 mM Tris-HCl 75 mM KCl 3 mM MgCl2 (pH 8.3 @ 25°C) Storage Buffer 20 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.01% IGEPAL® CA-630 pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes Unit Assay Conditions 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 6 mM MgCl 2 , 10 mM dithiothreitol, 0.01% IGEPAL CA-630, 0.5 mM dTTP, 0.4 mM poly(rA)•oligo(dT) 18 . FAQ Q: What is the difference between NEB# M0368 and NEB# M0253? A: M0368 is a mutant M-MuLV Reverse transcriptase with reduced RNase H activity and increased thermostability. The enzyme is active up to 50C, providing higher specificity, higher yield of cDNA, and more full-length cDNA product. Q: What is the optimal reaction temperature for ProtoScript II Reverse Transcriptase (M0368)? A: ProtoScript II Reverse Transcriptase is capable of generating cDNA of more than 10kb up to 48°C. We recommend 42°C for routine reverse transcription. Q: Can the cDNA products be used in real-time PCR analysis? A: Yes. The cDNA products generated by ProtoScript II Reverse Transcriptase can be used directly for real-time PCR analysis. Q: What thermostable DNA polymerase can be used for PCR after cDNA synthesis? A: For downstream PCR, we recommend OneTaq 2X Master Mix (NEB #M0482 or M0485) for PCR detection up to 5kb, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for highest fidelity, or LongAmp Taq 2X Master Mix (NEB #M0287) for high yields from longer products. Q: How can the yield be improved when using ProtoScript II Reverse Transcriptase? A: You can use higher amount of enzyme up to 1000U per 20ul cDNA synthesis reaction and increase the dNTP concentration up to 1mM. Q: How can the length of the product generated by M-MuLV Reverse Transcriptase be increased? A: Incubation at 42°C can relax some RNA secondary structure allowing M-MuLV Reverse Transcriptase to produce longer products. Q: Is RNaseH treatment required before PCR amplification? A: For most RT-PCR reactions, RNaseH treatment is not required. But for some difficult amplicons or sensitive assays, add 2U of E.coli RNaseH to the reaction and incubate at 37°C for 20min. Q: What is the difference between Induro Reverse Transcriptase (NEB #M0681) and ProtoScript II Reverse Transcriptase (NEB #M0368)? A: There are mainly two types of Reverse Transcriptases (RT): retrovirus-encoded RTs and intron-encoded RTs. Both RT types share some conserved domains and synthesize complementary DNA from an RNA template. The most common and well-characterized RTs are derived from retroviruses such as Moloney Murine Leukemia Virus (M-MuLV, MMLV), such as ProtoScript II Reverse Transcriptase. In contrast, Induro Reverse Transcriptase is a group II intron-encoded RT that exhibits higher thermostability, faster processivity, and better tolerance of reaction components when compared to retroviral RTs. Induro Reverse Transcriptase is useful for cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. Q: Why do I have low cDNA yields? A: There are several causes for low cDNA yield. Here are some suggestions to improve the yield: Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer (Agilent). Intact RNA of high purity is essential for full-length cDNA synthesis. RNA should have a minimum A260/A280 ratio of 1.7 or a RIN number greater than 8. Ethanol precipitation followed by a 70% ethanol wash can remove contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides. An RNA purification step using RNA extraction kits or phenol/chloroform extraction can remove contaminant proteins such as proteases. Increase the amount of RNA used, especially for low abundant RNA. Q: How do I know whether my template RNA is of good quality? A: Intact RNA of high purity is essential for full-length cDNA synthesis. An absorbance ratio at A260/A280 above 1.7 or RIN number greater than 8 on a BioAnalyzer usually indicates RNA of high quality.