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| Product Name | M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant, 200 U/uL |
| Catalog No. | IEC-HMM-0078 |
| Description | Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase engineered with point mutations in the RNase H domain that eliminate intrinsic RNase H activity while retaining full RNA-dependent and DNA-dependent DNA polymerase activity. The elimination of RNase H activity prevents degradation of the RNA template during first-strand cDNA synthesis, resulting in significantly longer cDNA products (up to 12 kb) and higher full-length cDNA yields compared to wild-type M-MLV RT. The enzyme is supplied at 200 U/uL and includes 5x reaction buffer and 0.1 M DTT. It is the preferred enzyme for cDNA synthesis for RT-PCR, qRT-PCR, cDNA library construction, 3'- and 5'-RACE, and RNA-seq library preparation requiring long transcripts. |
| Intended Use | First-strand cDNA synthesis from total RNA or poly(A)+ RNA for downstream applications including two-step RT-PCR, quantitative real-time RT-PCR (qRT-PCR), cDNA library construction, rapid amplification of cDNA ends (RACE), RNA sequencing (RNA-seq) library preparation, and probe synthesis for microarray and northern blot hybridization. |
| Principle / Technology | Engineered M-MLV reverse transcriptase catalyzes RNA-dependent DNA synthesis using an oligo(dT) primer, random hexamers, or gene-specific primer. The enzyme synthesizes a complementary DNA strand in the 5'->3' direction using RNA as template. The point mutations (D524N, etc.) in the RNase H domain abolish Mg2+-dependent hydrolysis of the RNA strand in RNA-DNA hybrids, preventing premature termination and 3' end degradation of the cDNA, leading to longer cDNA products and higher representation of 5' ends of long mRNAs. |
| Detection Method | Mix total RNA (1 pg-5 ug) or poly(A)+ RNA (1 pg-500 ng) with primer (oligo(dT)18 50 uM or random hexamers 50 ng/uL or gene-specific primer 2 pmol), dNTPs (0.5 mM each), 1x RT Buffer, 0.01 M DTT, 200 U M-MLV RT (RNase H-); incubate 50-60 min at 42 C (or 50 C for GC-rich templates with high secondary structure); inactivate 15 min at 70 C. |
| Sample Type | Total RNA (1 pg-5 ug in up to 10 uL), poly(A)+ mRNA (1 pg-500 ng), in vitro transcribed RNA, viral genomic RNA. |
| Performance Range / Specifications | cDNA synthesis length: up to 12 kb (first-strand); optimal synthesis 42 C (range 37-55 C); incorporation efficiency: >80% of RNA template converted to full-length cDNA for transcripts up to 5 kb; linear dynamic range: 1 pg-5 ug total RNA input. |
| Sensitivity / LOD | First-strand cDNA detectable from as little as 1 pg total RNA (approximately 1 cell equivalent) by qPCR of housekeeping gene (GAPDH, beta-actin); linear Cq vs log(input) over six orders of magnitude. |
| Specificity | RNase H- mutant specifically lacks RNase H activity while maintaining polymerase activity; no detectable RNase H activity in standard assay; sequence-nonspecific cDNA synthesis, primers determine specificity of initiation; no endogenous RNase, DNase, or nicking activity. |
| Reaction Conditions / Protocol | cDNA synthesis: 50-60 min at 42 C (optimal) or up to 55 C for GC-rich templates; heat inactivation: 15 min at 70 C; cDNA can be used directly for PCR (1-2 uL per 25 uL PCR reaction) or stored at -20 C. |
| Components / Formulation | M-MLV Reverse Transcriptase (RNase H-, 200 U/uL, in 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40, 50% glycerol), 5x RT Buffer (250 mM Tris-HCl pH 8.3 at 25 C, 375 mM KCl, 15 mM MgCl2), 0.1 M DTT, Protocol. |
| Storage Conditions | Store at -20 C; stable at 2-8 C for up to 1 month; avoid repeated freeze-thaw cycles. |
| Shelf Life | 24 months from date of manufacture at -20 C. |
| Package Specifications | 10,000 U (50 uL), 20,000 U (100 uL), 100,000 U (500 uL). |
| Product Form | Liquid; 200 U/uL in 50% glycerol storage buffer. |
| Quality Control | Each lot tested: no detectable RNase H activity (assay with [3H]-poly(A)-poly(dT) substrate); no detectable non-specific endonuclease or exonuclease activity; no detectable RNase contamination; full-length cDNA synthesis from 5 kb RNA transcript confirmed by agarose gel; functional qRT-PCR sensitivity down to 1 pg total RNA; no PCR inhibitors carried through. |
| Key Features | RNase H minus for longer cDNA (up to 12 kb); high sensitivity (1 pg RNA); functional at 37-55 C; active with oligo(dT), random hexamers, or gene-specific primers; no RNase H degradation of template RNA during synthesis. |
| Purity | >95% pure by SDS-PAGE; no detectable RNase H, RNase, DNase, endonuclease, or exonuclease activities; host genomic DNA <10 copies per unit. |
| Concentration | 200 U/uL (1 U = amount incorporating 1 nmol dTMP into acid-insoluble product in 10 min at 37 C using poly(A)-oligo(dT)12-18 as template-primer). |
| Activity / Unit Definition | Specific activity: >=50,000 U/mg protein; RNase H activity: not detectable (assay limit <0.01 U RNase H per 200 U RT). |
| Molecular Weight | Approximately 75 kDa (monomer). |
| Source / Origin | Recombinant M-MLV reverse transcriptase with point mutations in RNase H domain (D524N, etc.) expressed in E. coli; purified by multiple chromatography steps including affinity, ion exchange, and size exclusion; no animal-derived components. |
| pH Range / Optimal pH | Optimal pH 8.3 at 25 C (Tris-HCl); active range pH 7.5-9.0; 5x RT Buffer pH 8.3 at 25 C. |
| Shipping Conditions | Cold pack or dry ice; stable at ambient for up to 3 days during transit. |
| Expiration Date / Stability | 24 months at -20 C; 1 month at 2-8 C; quality retained after 15 freeze-thaw cycles. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. ISO 9001 certified manufacturing. |
| Compatibility | cDNA product compatible with all standard Taq and high-fidelity DNA polymerases for downstream PCR; compatible with SYBR Green and probe-based qPCR; cDNA may be purified by column or ethanol precipitation if buffer incompatibility is suspected. RNA template should be of high quality (RIN >=7 for best full-length synthesis). Inclusion of RNase inhibitor (e.g., RNasin) is recommended for long-term incubations or when working with low-abundance transcripts. |
| Recommended Buffer System | 5x RT Buffer: 250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2; enzyme storage: 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40, 50% glycerol. |
| Application Notes / Precautions | For GC-rich RNA templates with high secondary structure, increase reaction temperature to 50-55 C using gene-specific primers (oligo(dT) and random hexamers are less efficient at elevated temperatures). For long transcripts (>5 kb), use gene-specific primers and incubate at 42 C for 60-90 min. Include a no-RT control reaction (omit reverse transcriptase) for each RNA sample to detect genomic DNA contamination in downstream PCR. For qRT-PCR, dilute cDNA 1:5 to 1:20 in nuclease-free water before use. To preserve RNA, add RNase inhibitor (1 U/uL final) to all RT reactions. Store cDNA at -20 C (short term) or -80 C (long term). |
| Batch-to-Batch Consistency | cDNA yield from 1 ug HeLa total RNA within +/-20% of reference lot (GAPDH qPCR Cq); no RNase H activity detected for any lot; full-length cDNA synthesis confirmed for 5 kb control RNA. |
For research use only, not for clinical use.
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