Description
All-in-one RT master mix for qPCR
GenieScript All-in-One RT Master Mix for qPCR is a reverse transcription master mix that integrates genomic DNA removal and cDNA synthesis in a single tube. It uses a heat-labile DNase that is inactivated before reverse transcription, delivering gDNA-free cDNA for qPCR in around five minutes with minimal handling.
The 4X master mix contains a new-generation reverse transcriptase, heat-labile DNase (HL-DNase), RNase inhibitor, dNTPs and a random primer/Oligo (dT)20VN primer mix in an optimised buffer. Just add RNA template and water. The enzyme has high RNA affinity for efficient, consistent cDNA synthesis across a wide range of inputs, and performs well on GC-rich templates and complex secondary structures. The kit reverse-transcribes animal, plant and microbial RNA, and the cDNA is compatible with both dye-based and probe-based qPCR. For research use only.
Key Features
| True all-in-one workflow | gDNA removal and reverse transcription run in a single reaction with no separate DNase treatment. | |
| 5-minute reverse transcription | Takes RNA to qPCR-ready cDNA in as little as 5 minutes. | |
| Efficient gDNA removal | Integrated heat-labile DNase minimises genomic DNA contamination for reliable expression data. | |
| Handles challenging templates | Optimised for GC-rich RNA and complex secondary structures. | |
| Built for RT-qPCR | cDNA works with dye-based and probe-based qPCR for routine and high-throughput studies. |
Specifications
| Format | 4X all-in-one RT master mix (reverse transcriptase, HL-DNase, RNase inhibitor, dNTPs, primer mix) |
| Template | Total RNA, 1 pg - 1 μg per 20 μl reaction (up to 5 μg for low-expression targets) |
| Reaction time | 50°C for 5 min, 85°C for 5 sec |
| Priming | Random primers/Oligo (dT)20VN primer mix (included) |
| Downstream | Dye-based and probe-based qPCR |
| Reaction volume | 20 μl |
| Shelf life | 2 years |
Components
| Components | 100 rxns |
| RNase-free ddH2O | 2 × 1 ml |
| 4X GenieScript All-in-One RT Master Mix* | 500 μl |
*Contains reverse transcriptase, HL-DNase, RNase inhibitor, dNTP Mix and Random Primers/Oligo (dT)20VN Primer Mix.
Storage
Store at -30 ~ -15°C.
- Set up the reaction
- Mix the following in an RNase-free tube and pipette gently to mix thoroughly:
| Component | Volume |
| RNase-free ddH2O | To 20 μl |
| 4X GenieScript All-in-One RT Master Mix | 5 μl |
| Template RNA | Total RNA: 1 pg - 1 μg |
- Run the reaction
| Temperature | Time |
| 50°C | 5 min* |
| 85°C | 5 sec |
*Extend to 10 min if more cDNA is needed.
- Use or store the cDNA
- Use immediately for qPCR or store at -20°C for 6 months. For long-term storage, aliquot and store at -70°C. Avoid repeated freeze-thaw cycles.
Materials required
- RNase-free 1.5 ml centrifuge tubes, RNase-free 0.2 ml PCR tubes and RNase-free tips.
- Pipettes, PCR instrument, ice or ice box.
- A qPCR reagent: dye-based, e.g. GenieQ Universal SYBR qPCR Master Mix, or probe-based, e.g. GenieQ Universal Probe qPCR Master Mix.
Q1: How much RNA should I use?
- Use 1 pg - 1 μg total RNA per 20 μl reaction. For low-expression targets you can use up to 5 μg; more RNA than this pushes Ct values outside the linear range.
Q2: Do I need a separate DNase step?
- No. The master mix contains a heat-labile DNase that removes genomic DNA in the same tube and is inactivated before reverse transcription.
Q3: Can I use the cDNA for long-fragment PCR?
- Extending reverse transcription to 15 min gives cDNA up to 5 kb. For longer fragments, use the GenieScript IV 1st Strand cDNA Synthesis Kit.
Q4: How much cDNA can I add to a qPCR reaction?
- Undiluted cDNA should be no more than 1/10 of the qPCR reaction volume.
