OneScript® Hot 5X RT MasterMix
| Cat. No. | G590 | |||||||||
| Name | OneScript® Hot 5X RT MasterMix | |||||||||
| Unit | 100 rxn (400 µl) | |||||||||
| Category | Reverse Transcriptase & RT-PCR | |||||||||
| Description |
OneScript® Hot 5X RT MasterMix is a convenient, ready-to-use formulation designed for efficient and reliable first-strand cDNA synthesis. This optimized mix simplifies the workflow while ensuring exceptional reproducibility and performance. It contains abm’s proprietary OneScript® Hot Reverse Transcriptase, RNaseOFF Ribonuclease Inhibitor, dNTPs, and a precisely balanced combination of Oligo(dT)s and Random Primers. The OneScript® Hot Reverse Transcriptase features genetic modifications that abolish RNase H activity to achieve thermal stability, allowing the RNA template to remain intact during reverse transcription for improved cDNA yield and integrity. The resulting high-quality cDNA is ideal for a wide range of downstream molecular biology applications. Product Features:
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| Application |
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| Concentration | 5X | |||||||||
| Storage Condition |
Store at -20°C. |
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| Note |
If input RNA samples are expected to have high levels of genomic DNA contamination, we recommend using abm’s All-In-One 5X RT MasterMix with gDNA Removal (Cat. No. G592). |
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| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. G590 |
| Can I use both total RNA and poly(A)+ mRNA for cDNA synthesis? | |
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Yes, both total RNA and poly(A)+ mRNA can be used, though poly(A)+ mRNA typically yields higher quantities and better purity.
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| Can I synthesize cDNA from long RNA transcripts? | |
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Yes, for longer RNA transcripts, extend the 60°C incubation time to up to 30 minutes.
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| What can I use the synthesized cDNA for? | |
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The high-quality cDNA can be used in a variety of downstream applications, including gene expression analysis, cloning, and PCR-based assays. BlasTaq™ 2X qPCR Master Mix (Cat. No. G891) is well suited to downstream qPCR applications.
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| How should I store the synthesized cDNA? | |
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Store the synthesized first-strand cDNA at -20°C for long-term use.
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| Why am I getting low cDNA yields? | |
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Low cDNA yields can be caused by poor RNA integrity, contamination, or insufficient RNA input. To improve yield:
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| How much RNA template should I use for cDNA synthesis? | |
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We recommend using 1 ng to 2 μg of RNA per reaction.
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| What volumes of cDNA should I use for downstream PCR? | |
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Typically, use 1 μl of cDNA in a 25 μl PCR reaction. You can add up to 20% of the PCR volume (e.g., 5 μl in a 25 μl PCR), depending on your target and primers.
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What if input RNA samples are expected to have high levels of genomic DNA contamination?
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If input RNA samples are expected to have high levels of genomic DNA contamination, we recommend using abm’s All-In-One 5X RT MasterMix with gDNA Removal (Cat. No. G592).
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| What is the advantage of OneScript® Hot Reverse Transcriptase compared to OneScript® Plus Reverse Transcriptase? | |
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OneScript® Hot Reverse Transcriptase offers a significant advantage over enzymes that operate at lower temperatures (42-55ºC) due to its exceptional thermostability, allowing it to function effectively at 60-72ºC. This higher temperature improves efficiency by preventing RNA secondary structures and enabling cDNA synthesis from degraded or low-abundance RNA. Additionally, it is more resistant to inhibitors and contaminants, making it ideal for complex or impure RNA samples.
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Do I need an RNase inhibitor when using this product?
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All of abm's Reverse Transcriptases include RNaseOFF Ribonuclease Inhibitor, which protects RNA from degradation and is resistant to oxidation, even under low DTT concentrations. Therefore, there’s no need to add an external RNase inhibitor, as RNaseOFF provides optimal RNA protection during reverse transcription.
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| What discontinued abm products is Cat. No. G590 equivalent to? | |
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This product is abm’s next generation of RT MasterMixes and is functionally equivalent to G451, G452, G453, G454 (OneScript® cDNA Synthesis SuperMixes), with improved performance. Contact our customer service team technical@abmgood.com for more information.
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| What is the source of this enzyme? | |
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The enzyme is produced recombinantly in E. coli, which has been engineered to express the enzyme gene. While the original gene may come from another organism, all production and purification occur using E. coli under controlled conditions.
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| Can this product be used for next-generation sequencing (NGS) library preparation? | |
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This product can be used for NGS library preparation, but additional processing steps are recommended. NGS workflows require double-stranded DNA, and the presence of RNA:cDNA hybrids may interfere with downstream applications. To ensure compatibility, we suggest treating the reaction with RNase H to remove any residual RNA, followed by PCR amplification to generate the necessary double-stranded DNA for NGS library construction. |
| Is it normal to observe slight precipitation in the OneScript® Hot 5X RT MasterMix, and does it affect performance? | |
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Precipitation can occur during storage of the mastermix and does not affect reaction performance.
The precipitate can be readily reconstituted by gentle pipette mixing until the mastermix is homogenous. Once mixed, the mastermix will perform as expected. |