How long can I store the cDNA from my reverse transcription step?
You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.
Can I order the RNase Inhibitor from the High-Capacity cDNA Reverse Transcription Kit separately?
Yes. You can order the RNase Inhibitor separately as Cat. No. N8080119.
What type of primers are in the High-Capacity RNA-to-cDNA Kit?
The High Capacity RNA-to-cDNA Kit includes a 2X RT buffer mix, which is composed of dNTPs, random octamers, and oligo(dT)16.
When using the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919), I get high Ct values. What can I do?
It could be due to the following reasons:
- Insufficient cDNA template is present. Use 10 to 100 ng of cDNA template per 50 µL reaction.
- Quality of cDNA template is poor. Quantify the amount of cDNA template and test the cDNA template for the presence of PCR inhibitors.
- Sample degradation. Prepare fresh cDNA, then repeat the experiment.
- Reduced number of PCR cycles in the thermal cycling protocol. Increase the number of PCR cycles to the default setting of 40.
- Primer-dimer formation and residual polymerase activity. Prepare the reaction mixes and reaction plate on ice. To ensure optimal results, run the reaction plate as soon as possible after completing the reaction setup.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
When using the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919), the Multicomponent signal for ROX is not linear. What could cause this?
This could be due to the following reasons:
- Pure dye component’s spectra are incorrect. Rerun pure dye spectra.
- Incorrect dye components have been chosen. Choose correct dyes for data analysis.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.