CDNA synthesis is a vital step in genetic research and gene expression studies. With the growing interest in gene expression and molecular genetics, high-quality cDNA is in demand for various downstream applications like qPCR, cloning, and sequencing. A reliable cDNA synthesis kit can deliver precise, reproducible results.
The cDNA synthesis process involves converting RNA to cDNA, the complementary DNA strand. Reverse transcriptases catalyze this conversion in the presence of primers, dNTPs, and other reagents. Next, the resulting cDNA is amplified using PCR or qPCR methods. Here's a brief overview of the cDNA synthesis protocol and steps.
Materials Required for cDNA Synthesis
Before you start, prepare the following materials.
1. RNA sample
2. Oligo-dT or random hexamer primers
3. Reverse transcriptase enzyme
4. Buffer and DTT
5. dNTP mix
6. RNase inhibitor
7. PCR-grade water
cDNA Synthesis Steps
1. Add 1 µg of RNA to a reaction tube. The RNA sample should be of high quality, free from contaminants and degradation.
2. Add 1 µL of oligo-dT or random hexamer primers. The choice of primer depends on your experimental design.
3. Incubate the reaction mix at 65°C for 5 minutes. This helps to denature the RNA and anneal the primers.
4. Chill the reaction tube on ice for 2 minutes.
5. Add the following components in the order given below:
• Reverse transcriptase enzyme: 1 µL
• Buffer and DTT: 4 µL
• dNTP mix: 1 µL
• RNase inhibitor: 1 µL
• PCR-grade water: up to 20 µL
6. Mix the reaction tube, gently spin it, and incubate it at 42°C for 60 minutes. The optimal incubation time may vary depending on the reverse transcriptase enzyme used.
7. Inactivate the reverse transcriptase enzyme by heating the reaction tube at 70°C for 10 minutes.
8. Chill the reaction tube on ice for 2 minutes.
9. The cDNA obtained can be stored at -20°C for future use.
PCR Biosystems offers high-quality reverse transcriptase enzymes and cDNA synthesis kits suitable for a range of applications. Their thermostable reverse transcriptases ensure efficient cDNA synthesis, even from small or low-quality RNA samples. Additionally, their cDNA synthesis kits are optimized for performance and compatibility with downstream applications.
In conclusion, cDNA synthesis is a crucial step in genetic research and gene expression studies. High-quality cDNA can only be obtained using a reliable cDNA synthesis protocol and high-quality reagents. By following the protocol mentioned above and using trusted reagents, you can attain consistent and reproducible results for all your downstream applications.