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| Product Name | M-MLV Reverse Transcriptase (RNase H Minus) |
| Catalog No. | IEC-HMM-0051 |
| Description | A recombinant Moloney Murine Leukemia Virus reverse transcriptase engineered to eliminate RNase H activity through point mutations in the RNase H catalytic domain. The absence of RNase H activity prevents degradation of the RNA template during first-strand cDNA synthesis, resulting in longer cDNA products and higher yields compared to wild-type enzyme. |
| Intended Use | For first-strand cDNA synthesis from total RNA or mRNA templates in two-step RT-PCR, cDNA library construction, and RNA sequencing library preparation. |
| Principle / Technology | RNA-dependent DNA polymerase activity synthesizes a complementary DNA strand from an RNA template using oligo-dT, random hexamer, or gene-specific primers. The engineered enzyme lacks RNase H activity, so the RNA template remains intact throughout synthesis, enabling full-length cDNA production from long transcripts. |
| Detection Method | cDNA synthesis by reverse transcription monitored by incorporation of radiolabeled dNTPs. Product length distribution assessed by alkaline agarose gel electrophoresis. |
| Sample Type | Total RNA (1 pg to 5 ug) or poly(A)+ mRNA extracted from cultured cells, animal tissues, plant materials, or microorganisms. |
| Performance Range / Specifications | Synthesizes first-strand cDNA up to 12 kb in length. Linear relationship between input RNA and cDNA yield across 1 pg to 5 ug input range. |
| Sensitivity / LOD | Detects transcripts from as little as 1 pg total RNA. Suitable for single-cell RNA applications when combined with amplification strategies. |
| Specificity | High specificity for RNA templates; no detectable DNA-dependent DNA polymerase activity. Enzyme does not utilize DNA as a template. |
| Reaction Conditions / Protocol | Template RNA (1 pg - 5 ug) + primer + dNTPs, denature at 65 °C 5 min, chill on ice. Add enzyme + buffer, incubate at 42 °C 30-60 min. Inactivate at 70 °C 15 min. Use 2-5 uL of resulting cDNA for downstream PCR. |
| 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, 50% glycerol. Supplied with 5x Reaction Buffer and 100 mM DTT. |
| Storage Conditions | Store at -20 °C to -30 °C. Aliquot on first use to avoid repeated freeze-thaw cycles. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 10,000 U (50 uL), 50,000 U (250 uL), 200,000 U (1 mL). Includes 5x RT Buffer and 100 mM DTT solution. |
| Product Form | Clear, colorless liquid in glycerol-based storage buffer. |
| Quality Control | Each lot tested for: RT specific activity; absence of contaminating DNase, RNase, and endonuclease; functional cDNA synthesis from 1 pg - 5 ug control RNA with reproducible Ct values by qPCR; absence of detectable RNase H activity. |
| Key Features | Engineered RNase H- mutation for full-length cDNA synthesis; high thermostability (active up to 50 °C); generates cDNA up to 12 kb; compatible with downstream PCR, qPCR, and library preparation workflows. |
| Purity | >= 95% by SDS-PAGE. No detectable E. coli genomic DNA or host protein contamination by sensitive methods. |
| Concentration | 200 U/uL standard. Higher concentration variants (400 U/uL) available for specialized applications. |
| Activity / Unit Definition | 1 unit incorporates 1 nmol dTMP into acid-insoluble product in 10 min at 37 °C using poly(A)·oligo(dT)₁₂₋₁₈ as template-primer. |
| Molecular Weight | ~75 kDa (monomer, processed form). |
| Source / Origin | Recombinant enzyme expressed in E. coli. Gene derived from Moloney Murine Leukemia Virus with engineered point mutations in the RNase H domain to eliminate hydrolytic activity. |
| pH Range / Optimal pH | Optimal activity at pH 7.5-8.3 (Tris-HCl buffer). Retains >50% activity from pH 7.0-8.8. |
| Shipping Conditions | Shipped with cold packs to maintain below 10 °C. Stable during transit for up to 72 hours at ambient temperature if promptly frozen upon receipt. |
| Expiration Date / Stability | 24 months at -20 °C. Minimize exposure to room temperature during use; keep on ice at all times when not frozen. |
| Regulatory / Compliance | ISO 9001 certified manufacturing. Suitable as IVD raw material for diagnostic kit development. Animal-origin-free manufacturing process available for specific applications. |
| Compatibility | Compatible with downstream Taq-based PCR, high-fidelity PCR, qPCR, and NGS library preparation. Produces cDNA suitable for cloning and Sanger sequencing. |
| Recommended Buffer System | 5x Reaction Buffer: 250 mM Tris-HCl (pH 8.3 at 25 °C), 375 mM KCl, 15 mM MgCl₂. DTT added separately to a final concentration of 10 mM in the reaction. |
| Application Notes / Precautions | For GC-rich or highly structured RNA, perform the reverse transcription at 45-50 °C for improved strand displacement. For very long transcripts (>8 kb), extend the incubation to 60-90 min. Always include a no-RT control to verify the absence of genomic DNA contamination. |
| Batch-to-Batch Consistency | Lot-to-lot activity variation <= +/- 10%. Functional validation includes standardized cDNA synthesis and qPCR testing across multiple RNA input concentrations. |
For research use only, not for clinical use.
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