SuperScript IV UniPrime One-Step RT-PCR System, 100 reactions - FAQs

Yes, reaction setup with SuperScript IV UniPrime One-Step RT-PCR System can be performed at room temperature. Moreover, the innovative two-phase hot start mechanism allows preassembled reactions to remain stable at room temperature for up to 24 hrs for targets up to 3 kb and for up to 4 hrs for targets >3 kb.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

SuperScript IV UniPrime One-Step RT-PCR System includes 2X UniPrime RT-PCR master mix containing modified Platinum SuperFi II DNA Polymerase, which provides high specificity and high yields, and is ideally suited for applications that require sequence accuracy. On the contrary to common proofreading enzymes, this polymerase is compatible with dUTP.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

Red and blue tracking dyes included in the colored format of SuperScript IV UniPrime One-Step RT-PCR System do not interfere with electrophoresis in E-Gel agarose gels. Please note that the reaction product obtained with both the colored and dye-free format should be diluted 2- to 30-fold for optimal separation using E-Gel agarose gels.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

The two-phase hot-start mechanism, utilized in the SuperScript IV One-Step RT-PCR System and SuperScript IV UniPrime One-Step RT-PCR System, ensures sequential activation of RT and PCR enzymes in the one-step RT-PCR workflow. At ambient temperature, SuperScript IV RT is maintained inactive with a heat-sensitive RT-blocker. During the first hot-start activation phase at approximately 45 degrees C, the RT-blocker is released and the first-strand cDNA synthesis is initiated. During the second activation phase, the reaction is heated to 98 degrees C to activate DNA Polymerase and simultaneously inactivate SuperScript IV RT. This mechanism separates the RT and PCR enzymes' activities, delivering the highest RT-PCR specificity and yield.

Good laboratory practices are important for long fragment, one-step RT-PCR. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Optimization steps to consider include use of longer extension times and increasing template amounts. Learn more about RT-PCR reaction optimization and setup by visiting our reverse transcription educational resources (https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education.html).