RT Master Mix for qPCR III
Based on 1 publication(s) in Google Scholar
MCE MCE Reverse Transcription Kit III is suitable for the conventional synthesis of cDNA from RNA and the stem-loop method for the synthesis of cDNA from microRNA (miRNA).
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Storage :
-20℃，2 years
Protect from light and avoid repeated freeze-thaw cycles
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Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
MCE MCE Reverse Transcription Kit III is suitable for the conventional synthesis of cDNA from RNA and the stem-loop method for the synthesis of cDNA from microRNA (miRNA). The kit contains third-generation M-MLV reverse transcriptase and various components required for reverse transcription to synthesize cDNA. Simply add the RNA template, primers, and water to start the reaction. Different types of reverse transcription primers can be flexibly used depending on the experimental design and up to 12 kb of cDNA can be obtained within 15 min.
Upon completion of the first-strand cDNA synthesis, the cDNA product can be directly applied as a template in a standard PCR and qPCR. MCE SYBR Green qPCR Master Mix (HY-K0501, HY-K0501A) is highly recommended for detection of the expression levels of interested genes.
Protocol
1. Removal of genomic DNA from RNA (optional)
| Component | Volume |
|---|---|
| 10× dsDNase Buffer | 2 μL |
| Total RNA | X μL (recommended ≤1 μg) |
| dsDNase | 1 μL |
| Nuclease-Free H2O | To 10 μL |
Mix gently, briefly centrifuge, and incubate at 37°C for 2 min. The treated RNA may be used directly for reverse transcription.
2. First-strand cDNA synthesis
Select Oligo(dT)20VN, Random hexamers, or a gene-specific RT primer according to the experimental design. Prepare the reaction on ice:
| Component | 20 μL reaction |
|---|---|
| RNA RT Mix | 4 μL |
| Oligo(dT)20VN (50 μM) and/or Random hexamers (50 μM) | Add as required |
| gDNA-depleted RNA mixture / RNA template | X μL |
| Nuclease-Free H2O | To 20 μL |
Note: The recommended amount of RNA RT Mix is 4 μL per 20 μL reaction. Adjust RNA template and primer volumes according to the experimental design and bring to the final volume with RNase-Free H2O.
Run the following program:
| Step | Temperature | Time |
|---|---|---|
| Primer annealing | 25°C | 5 min |
| Reverse transcription | 55°C | 15 min |
| Enzyme inactivation | 85°C | 2 min |
| Hold | 4°C | Hold |
The resulting cDNA may be used directly as template for conventional PCR or qPCR. Dilute the cDNA as appropriate for qPCR according to target abundance.
Publications
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Journal Impact Factor
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Storage
-20℃，2 years
Protect from light and avoid repeated freeze-thaw cycles
Components
| Components | HY-K0513-100 rxns |
|---|---|
| RNA RT Mix | 400 μL |
| 10× dsDNase Buffer | 200 μL |
| Oligo(dT)20VN (50 μM) | 100 μL |
| Random hexamers (50 μM) | 100 μL |
| dsDNase | 50 μL × 2 |
| Nuclease-Free H2O | 1 mL × 2 |
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