Description
First-strand cDNA synthesis kit
GenieScript IV 1st Strand cDNA Synthesis Kit is a flexible first-strand cDNA synthesis kit built on a 4th-generation reverse transcriptase. It supports oligo(dT), random or gene-specific priming for qPCR, cloning and long-cDNA applications. High thermostability and inhibitor tolerance give efficient, full-length cDNA from challenging or low-input RNA, with a separate genomic DNA removal step.
The 4X GenieScript IV RT Master Mix premixes the reverse transcriptase, RNase inhibitor and dNTPs, so you only add the primers suited to your downstream experiment. Compared with earlier versions, the enzyme offers stronger extension, faster reactions and higher inhibitor tolerance, and synthesises up to 15 kb cDNA in 5 minutes. The 5X gDNA Removal Mix removes genomic DNA contamination in 2 min at 42°C. The kit uses total RNA or poly(A)+ RNA from animal, plant and microbial sources, and is especially suited to qPCR quantification and long-fragment cDNA amplification. For research use only.
Key Features
| Longer cDNA | Synthesises cDNA up to 15 kb. | |
| Higher RT efficiency | Improved reverse transcription efficiency gives lower Ct values. | |
| Fast reaction | Efficient reverse transcription in 5 minutes. | |
| Rapid gDNA removal | Genomic DNA is eliminated in 2 min at 42°C. | |
| Convenient premix | RT master mix contains enzyme, RNase inhibitor and dNTPs; just add primers. |
Specifications
| Template | Total RNA (10 pg - 5 μg) or poly(A)+ RNA (10 pg - 500 ng) from animal, plant and microbial sources |
| Maximum cDNA length | 15 kb |
| Reaction time | 5 min at 50°C (PCR); 15 min at 37°C (qPCR) |
| gDNA removal | 42°C, 2 min |
| Priming | Oligo(dT)20VN, random primers or gene-specific primers |
| Reaction volume | 20 μl |
| Shelf life | 2 years |
Components
| Components | 50 rxns | 100 rxns |
| RNase-free ddH2O | 1 ml | 1 ml |
| 5X gDNA Removal Mix | 100 μl | 200 μl |
| 4X GenieScript IV RT Master Mix* | 250 μl | 500 μl |
| Oligo (dT)20VN | 50 μl | 100 μl |
| Random Primers | 100 μl | 200 μl |
| 4X No RT Control Mix | 25 μl | 50 μl |
*Contains dNTP Mix and RNase inhibitor. Reaction volume is 20 μl/rxn.
Storage
Store at -30 ~ -15°C.
A. cDNA synthesis for PCR
- Denature the RNA
| Component | Volume / amount |
| RNase-free ddH2O | To 8 μl |
| Total RNA | 10 pg - 5 μg |
| or Poly(A)+ RNA | 10 pg - 500 ng |
- Incubate at 65°C for 5 min, then chill on ice immediately for 2 min.
- Denaturation opens secondary structures and improves first-strand yield. Do not skip it for cDNA longer than 3 kb.
- Intact RNA is essential for long cDNA. If integrity is uncertain, increase RNA input (up to 5 μg) to raise the number of intact copies.
- Remove genomic DNA
| Component | Volume |
| Mixture from step 1 | 8 μl |
| 5X gDNA Removal Mix | 2 μl |
- Pipette gently to mix. Incubate at 42°C for 2 min.
- Set up the first-strand reaction
| Component | Volume |
| Mixture from step 2 | 10 μl |
| 4X GenieScript IV RT Master Mix | 5 μl |
| Oligo (dT)20VN* | 1 μl |
| RNase-free ddH2O | 4 μl |
*Alternatively, use a gene-specific primer (2 pmol) or Random Primers. Pipette gently to mix.
- Run the reaction
| Temperature | Time |
| 50°C | 5 min* |
| 85°C | 5 sec |
*15 kb cDNA is synthesised within 5 min. Extend to 30 min if more cDNA is needed.
B. cDNA synthesis for qPCR
- Remove genomic DNA
| Component | Volume / amount |
| RNase-free ddH2O | To 10 μl |
| 5X gDNA Removal Mix | 2 μl |
| Total RNA | 10 pg - 1 μg |
| or Poly(A)+ RNA | 10 pg - 100 ng |
- Pipette gently to mix. Incubate at 42°C for 2 min.
- Set up the first-strand reaction
| Component | Volume |
| Mixture from step 1 | 10 μl |
| 4X GenieScript IV RT Master Mix | 5 μl |
| Oligo (dT)20VN | 1 μl |
| Random Primers | 2 μl |
| RNase-free ddH2O | 2 μl |
Alternatively, use a gene-specific primer (2 pmol). Pipette gently to mix.
- Run the reaction
| Temperature | Time |
| 37°C* | 15 min |
| 85°C | 5 sec |
*For templates with complex secondary structure or high GC content, raise the temperature to 50°C to improve yield.
C. No RT control (optional)
A negative control without reverse transcriptase tests for residual genomic DNA in the RNA template and confirms gDNA removal.
| Component | Volume |
| gDNA-removed mixture* | 10 μl |
| 4X No RT Control Mix | 5 μl |
| RNase-free ddH2O | 5 μl |
*The mixture at the end of step 2 (PCR workflow) or step 1 (qPCR workflow). Pipette gently to mix, then incubate at 50°C (for PCR) or 37°C (for qPCR) for 15 min, followed by 85°C for 5 sec.
cDNA storage
- Use cDNA immediately or store at -20°C for 6 months. For long-term storage, aliquot and store at -70°C. Avoid repeated freeze-thaw cycles.
Q1: Which primer should I use for cDNA synthesis?
- For PCR from eukaryotic RNA, Oligo (dT)20VN gives the highest yield of full-length cDNA; gene-specific primers give the highest specificity; random primers suit GC-rich or structured regions and prokaryotic templates. For qPCR, use the recommended mix of Oligo (dT)20VN and Random Primers for even coverage of the mRNA.
Q2: Can I skip the genomic DNA removal step?
- Yes. Reverse transcription can be performed directly; make up the missing volume with RNase-free ddH2O.
Q3: How can I improve yield of long cDNA fragments?
- Do not skip the 65°C denaturation step for cDNA longer than 3 kb, use intact RNA, increase RNA input up to 5 μg if integrity is uncertain, and extend the 50°C reaction to 30 min if more cDNA is needed.
Q4: What is the No RT Control Mix for?
- It sets up a negative control reaction without reverse transcriptase to test for residual genomic DNA in the RNA and confirm that gDNA removal worked.
Q5: How should I store the cDNA?
- Use it immediately or store at -20°C for 6 months. For long-term storage, aliquot and keep at -70°C, and avoid repeated freeze-thaw cycles.
