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.
Which reverse transcription kit can I use with the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919)?
We recommend using the High-Capacity RNA-to-cDNA Kit (Cat. No. 4387406, 4388950).
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
What primer concentrations can I use with the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919)?
The recommended primer concentration range is 300-800 nM. You will need to optimize the primer concentration by running different concentration combinations of the forward and reverse primer. See page 28 of the PowerUp SYBR Green Master Mix User Guide.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
Does the PowerUp SYBR Green Master Mix (Cat. No. A25741, A25742, A25743, A25776, A25777, A25778, A25779, A25780, A25919) contain uracil-DNA glycolase?
This master mix has a heat-labile UDG. It is completely and irreversibly inactivated after 10 minute incubation at 50°C.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.