PowerUp™ SYBR™ Green Master Mix for qPCR, 2 x 5 mL - FAQs

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.

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.

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.

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.

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.

The PowerUp SYBR Green Master Mix uses a Dual Lock Taq DNA Polymerase enzyme. This enzyme combines two unique hot-start mechanisms to help control its activity and to prevent undesirable early activity of the polymerase at low temperatures. As such, this mechanism provides excellent specificity.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

We have not tested PowerUp SYBR Green Master Mix for qPCR at -20 degrees C. We have only performed accelerated and real-time stability testing at 2-8 degrees C, as this is the recommended and most convenient storage temperature.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

The recommended storage temperature for the PowerUp SYBR Green Master Mix is 2-8 degrees C.

Both master mixes are compatible with all Applied Biosystems Real Time PCR instruments, but Power SYBR Green Master Mix can only be run in Standard Mode. PowerUp SYBR Green Master Mix contains a heat-labile UDG, which allows for review of PCR products up to 72 hrs post-PCR (unlike Power SYBR Green Master Mix). Finally, PowerUp SYBR Green Master Mix uses Dual-Lock Taq DNA Polymerase, which uses a combination of two hot start modifications for exceptional specificity.

PowerUp SYBR Green Master Mix can be used in either standard or fast cycling mode and is compatible with all Applied Biosystems Real-time PCR instruments (except the 7900HT Fast mode instrument). It is also compatible with the Bio-Rad iQ5, Roche LightCycler 480, and Agilent MX3005P systems.