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| Product Name | AMV Reverse Transcriptase |
| Catalog No. | IEC-HMM-0052 |
| Description | Reverse transcriptase purified from Avian Myeloblastosis Virus with inherent RNase H activity. This robust enzyme operates at a higher optimal temperature (42-50 °C) than M-MLV RT, which helps resolve secondary structures in GC-rich or highly structured RNA templates, enabling more complete reverse transcription of challenging templates. |
| Intended Use | For cDNA synthesis from difficult RNA templates with extensive secondary structure, particularly useful for GC-rich transcripts, viral RNA genomes, and templates where elevated reaction temperatures are beneficial for specificity. |
| Principle / Technology | RNA-dependent DNA polymerase catalyzes synthesis of complementary DNA from an RNA template using a primer with a free 3'-hydroxyl group. The associated RNase H activity degrades the RNA strand of RNA-DNA hybrids, which can facilitate second-strand synthesis but may reduce full-length product yields for long transcripts. |
| Detection Method | Activity measured by incorporation of [³H]-dTMP into acid-insoluble product using poly(A)·oligo(dT) as template-primer at 37 °C. |
| Sample Type | Total RNA or mRNA from any biological source; especially effective for plant RNA, viral RNA, and GC-rich transcripts with significant secondary structure. |
| Performance Range / Specifications | Active at 37-55 °C; optimal 42-50 °C. cDNA products typically 500 bp to 5 kb. Higher temperature operation improves specificity for challenging templates. |
| Sensitivity / LOD | Detectable cDNA synthesis from as little as 10 pg total RNA input under optimal conditions. |
| Specificity | Higher reaction temperature (42-50 °C) reduces non-specific priming compared to mesophilic RT enzymes. Inherent RNase H activity may truncate very long cDNA products. |
| Reaction Conditions / Protocol | Template RNA + primer (oligo-dT, random hexamer, or gene-specific) + dNTPs, 65 °C 5 min, chill on ice. Add enzyme + buffer, incubate at 42-50 °C 30-60 min. Inactivate at 85 °C 5 min. |
| Components / Formulation | AMV Reverse Transcriptase (10 U/uL) in 200 mM potassium phosphate (pH 7.2), 2 mM DTT, 0.2% Triton X-100, 50% glycerol. Supplied with 5x AMV Reaction Buffer. |
| Storage Conditions | Store at -20 °C to -30 °C. Do not expose to temperatures above 4 °C for extended periods. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 500 U (50 uL), 2,500 U (250 uL), 10,000 U (1 mL). Includes 5x AMV Buffer. |
| Product Form | Clear, colorless solution containing glycerol as cryoprotectant. |
| Quality Control | Each lot verified for: specific activity (>= 40,000 U/mg); absence of contaminating DNase and non-specific endonuclease; functional cDNA synthesis performance at 42 °C and 50 °C; qPCR testing of cDNA products. |
| Key Features | Tolerates elevated reaction temperatures (up to 55 °C) for structured RNA resolution; robust performance with GC-rich templates; well-established enzyme with decades of published validation. |
| Purity | >= 90% by SDS-PAGE. Purified from native viral source or recombinant expression depending on manufacturing route. |
| Concentration | 10 U/uL standard formulation. |
| Activity / Unit Definition | 1 unit incorporates 1 nmol dTMP into acid-insoluble product in 10 min at 37 °C using poly(A)·oligo(dT) template-primer. |
| Molecular Weight | Heterodimer: alpha subunit ~63 kDa, beta subunit ~95 kDa. |
| Source / Origin | Native enzyme purified from Avian Myeloblastosis Virus (AMV) particles or produced as recombinant protein in E. coli or baculovirus expression systems. |
| pH Range / Optimal pH | Optimal pH 8.0-8.5. Active range pH 7.5-9.0. |
| Shipping Conditions | Shipped with cold packs to maintain below 10 °C. |
| Expiration Date / Stability | 24 months at -20 °C. Stable on ice for up to 8 hours during protocol setup. |
| Regulatory / Compliance | ISO 9001 certified production. For research use and IVD raw material supply. Regulatory documentation available on request. |
| Compatibility | Compatible with all common cDNA synthesis primers and downstream PCR applications. Produces cDNA suitable for both endpoint and real-time PCR. |
| Recommended Buffer System | 5x AMV Buffer: 250 mM Tris-HCl (pH 8.3), 250 mM KCl, 50 mM MgCl₂, 2.5 mM spermidine, 50 mM DTT. |
| Application Notes / Precautions | For highly structured RNA, extend the denaturation step to 10 min at 70 °C before adding enzyme. Reaction temperature of 50 °C yields the best balance of activity and specificity for most challenging templates. RNase inhibitor supplementation recommended for precious RNA samples. |
| Batch-to-Batch Consistency | Activity variation between lots <= +/- 15%. Each lot tested against standardized RNA templates for consistent cDNA yield. |
For research use only, not for clinical use.
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