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Thermo Scientific™ RevertAid H Minus Reverse Transcriptase (200 U/μL)
Description
Thermo Scientific RevertAid H Minus Reverse Transcriptase is a recombinant M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity, but lacks RNase H activity due to a point mutation in the RNase H domain. RevertAid H Minus Reverse Transcriptase does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA, and therefore high yields of full-length cDNA from long templates are obtained. RevertAid H Minus Reverse Transcriptase maintains activity over a wide temperature range (42–50°C).
Features of RevertAid H Minus Reverse Transcriptase include:
• High yields of full-length first strand cDNA up to 13 kb
• Optimum activity at 42–45°C
• Active up to 55°C
• Incorporates modified nucleotides (e.g., Cy3-, Cy5-, rhodamine-, aminoallyl-, and fluorescein-labeled nucleotides)
Applications
• First strand cDNA synthesis for RT-PCR and real-time RT-PCR
• Reverse transcription at elevated temperatures to reduce effects of secondary structure
• Synthesis of cDNA for cloning and expression
• Generation of labeled cDNA probes for microarrays
• DNA labeling
• Analysis of RNA by primer extension
Specifications
Specifications
| Concentration | 200 U/μL |
| Content And Storage | • RevertAid H Minus Reverse Transcriptase (200 U/μL) |
| Format | Stand-alone Enzyme |
| Reaction Speed | 60 min. |
| Technique | Reverse Transcription |
| Optimal Reaction Temperature | 42°C to 45°C |
| Quantity | 5 x 10,000 Units |
| Reverse Transcriptase | RevertAid H Minus |
| Ribonuclease H Activity | Reduced |
| Shipping Condition | Dry Ice |
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Frequently Asked Questions (FAQs)
It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H-minus RTs for template-independent addition of C nucleotides.
Trace amounts of reagents used in RNA purification protocols may remain in solution and inhibit first-strand synthesis, e.g., SDS, EDTA, guanidine salts, phosphate, pyrophosphate, polyamines, spermidine. To remove trace contaminants, we recommend re-precipitating the RNA with ethanol and washing the pellet with 75% ethanol, or re-purifying the RNA.
RNA purity and integrity are essential for synthesis and quantification of cDNA. Always assess the integrity of RNA prior to cDNA synthesis. Use freshly prepared RNA. Multiple freeze/thaw cycles of the RNA sample and synthesized cDNA is not recommended. Avoid RNase contamination and discard low quality RNA.
It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H Minus RTs for template-independent addition of C nucleotides. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in 1-step RT-qPCR applications.
All Thermo Scientific reverse transcriptases (RevertAid RT, RevertAid H Minus RT, Maxima RT, and Maxima H Minus RT) possess intrinsic TdT activity, although at varying degrees depending upon the reaction conditions. We recommend specialized SuperScript IV Template Switching RT Master Mix for high efficiency in applications requiring template switching RT.
For Research Use Only. Not for use in diagnostic procedures.