Description
Product description
This product is a classic one-step cDNA synthesis reverse transcription ready-to-use premix from an RNA template. The core reverse transcriptase contained in the product has been effectively engineered to exhibit excellent thermal stability and template affinity, allowing it to tolerate reaction temperatures as high as 60°C. It is suitable for reverse transcription of RNA templates with complex secondary structures, low amounts of template, and low-copy genes. Additionally, this product contains a gDNA digestion component that can remove residual genomic DNA contamination from the RNA template, ensuring more reliable downstream results.
The cDNA synthesized by this product is suitable for downstream qPCR applications. It is recommended to use it in combination with the Universal Multiplex qPCR Probe Premix (Cat#N132041), Universal qPCR Dye Premix (Cat#N132031), or the Traceable qPCR Dye Premix (Cat#N132034, Cat#N132035, Cat#N132036) for high-performance gene expression analysis.
Specifications
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Catalog Number
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N132061E
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N132061S
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|
Specifications
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10 T
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100 T
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Components
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Catalog Number
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Component Name
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N132061E
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N132061S
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N132061-A
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RNase-free H2O
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1 mL
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2×1 mL
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N132061-B
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5×gDNA Digester Mix
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30 μL
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320 μL
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N132061-C
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4×cDNA Synthesis SuperMix
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50 μL
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500 μL
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【Note】Component C of the 4×cDNA Synthesis SuperMix contains the gDNA Digester terminator.
Storage
Transport with ice packs. Store at -20℃. Valid for 18 months.
Notes
1. Component B (5× gDNA Digester Mix) and Component C (4× cDNA Synthesis SuperMix) both contain high concentrations of glycerol. Before use, briefly centrifuge to collect the components at the bottom of the reaction tube. Then gently mix thoroughly by pipetting up and down, and accurately aspirate the required volume.
2. If the volume of the RNA template to be added is relatively large (e.g., more than 2 μL), ensure that the RNA is dissolved in water rather than TE buffer, as TE buffer may inhibit gDNA removal and reverse transcription.
3. The gDNA removal step can be omitted, and reverse transcription can be performed directly using Component C (4× cDNA Synthesis SuperMix).
4. The preparation of the reaction mixture should be carried out on ice to maintain stability. During the process, avoid contamination with RNase.
5. For your safety and health, please wear a lab coat and disposable gloves when operating.
6. For Research Use Only.
Instructions
1. Removal of Residual Genomic DNA
Prepare the following mixture in an RNase-free centrifuge tube and gently mix by pipetting. Incubate at 42°C for 2 minutes.
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Component
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Usage Amount
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RNase Free H2O
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To 15 μL
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5×gDNA Digester Mix
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3 μL
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Total RNA or mRNA
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RNA：10 pg-5 μg
mRNA：10 pg-500 ng
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【Note】For a 20 μL reverse transcription reaction system, it is recommended that the input amount of Total RNA does not exceed 1 μg. If the expression level of the target gene is low, up to 5 μg of Total RNA can be used. However, excessive RNA input may exceed the linear range of subsequent quantitative PCR.
2. Reverse Transcription Reaction Setup
Directly add the 4× cDNA Synthesis SuperMix into the reaction tube from Step 1 and gently mix by pipetting.
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Component
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Usage Amount
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Step 1 Reaction Mixture
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15 μL
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4×cDNA Synthesis SuperMix
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5 μL
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3. Reverse Transcription Procedure Settings
1) Standard procedure
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Temperature
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Time
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25℃
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5 min
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55℃
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15 min
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85℃
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5 min
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【Note】The recommended reverse transcription temperature is 55℃. For templates with high GC content or complex structures, the reverse transcription temperature can be increased to 60℃.
2) Rapid Program [Suitable for templates with GC content ≤ 55% or simple templates]
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Temperature
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Time
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55℃
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15 min
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85℃
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5 sec
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【Note】The reverse transcription product can be used immediately for qPCR reactions or stored short-term at -20°C. For long-term storage, it is recommended to aliquot and store at -80°C to avoid repeated freeze-thaw cycles.
