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View additional product information for SuperScript™ IV One-Step RT-PCR System - FAQs (12594100, 12595100)
15 product FAQs found
The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50°C). The additional inactivation step is therefore not required in RT-qPCR applications.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
The only change is that the incubation time for the reverse transcription reaction has been reduced from 50 minutes to 10 minutes. All the other parameters and steps are the same.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
We have not created a one-step RT-qPCR kit containing SuperScript IV RT for real-time PCR. However, it is included in end-point one-step RT-PCR kits: https://thermofisher.com/ssiv-onestep
We also offer a two-step RT-qPCR kit containing SuperScript IV RT: https://www.thermofisher.com/vilo
For one-step RT-qPCR, we offer a kit with SuperScript III RT: https://www.thermofisher.com/order/catalog/product/117320200
Due to the high processivity of SuperScript IV RT and Platinum SuperFi DNA Polymerase, the SuperScript IV One-Step RT-PCR System enables detection of a broad range of target lengths, from 0.2 to 13.8 kb.
The minimum amount of total RNA required for the SuperScript IV One-Step RT-PCR System is 0.01 pg.
Yes, preassembled reactions set up using the SuperScript IV One-Step RT-PCR System can be left at room temperature for up to 4 hours before cycling.
Yes, with the SuperScript IV One-Step RT-PCR System, we have had success amplifying up to 4 targets in the same reaction.
Platinum SuperFi DNA Polymerase in the SuperScript IV One-Step RT-PCR System produces blunt-end PCR products that can be cloned directly into blunt-end cloning vectors. For TA cloning, 3′ dA-overhangs would have to be added to the PCR product.
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.
With the SuperScript IV One-Step RT-PCR System, cDNA synthesis can be performed at higher temperatures than with other one-step RT-PCR products. For GC-rich or structurally complex RNA templates, we recommend increasing the cDNA synthesis incubation temperatures up to 55-60 degrees C.
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).
The RT-PCR cycling conditions with the SuperScript IV One-Step RT-PCR System differ significantly from other one-step RT-PCR products. For the best results, we recommend using the cycling conditions described in the SuperScript IV One-Step RT-PCR System user guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0016518_superscriptIV_1step_master_mix_UG.pdf).
The optimal annealing temperature for your primers may differ significantly when using the SuperScript IV One-Step RT-PCR System in comparison to other one-step RT-PCR products, due to the difference in buffer salt concentration. We recommend using the Tm calculator (thermofisher.com/tmcalculator) to determine your primers' Tm values and suggested annealing temperature.