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LIFE TECHNOLOGIES SS IV 1-STEP RT-PCR SYS 25 RXN

Catalog No. 12594025
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Catalog No. 12-594-025 Supplier LIFE TECHNOLOGIES Supplier No. 12594025

Frequently Asked Questions (FAQs)

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

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 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix. 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.
Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.
Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

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).
Do you offer a one-step RT-qPCR kit containing SuperScript IV RT?

SuperScript IV RT is not recommended for one-step RT-qPCR applications. However, it is included in end-point one-step RT-PCR kits: https://thermofisher.com/ssiv-onestep

For one-step RT-qPCR, we recommend SuperScript III Flash Reverse Transcriptase or TaqPath RT-qPCR Master Mix products.
What is the largest target size that can be amplified with the SuperScript IV One-Step RT-PCR System?

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.
What is the minimum amount of total RNA required for the SuperScript IV One-Step RT-PCR System?

The minimum amount of total RNA required for the SuperScript IV One-Step RT-PCR System is 0.01 pg.
Can I assemble the reaction ahead of time when using the SuperScript IV One-Step RT-PCR System?

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.
Can I multiplex with the SuperScript IV One-Step RT-PCR System?

Yes, with the SuperScript IV One-Step RT-PCR System, we have had success amplifying up to 4 targets in the same reaction. For enhanced multiplexing capabilities, we recommend using SuperScript IV UniPrime One-Step RT-PCR System, which can help improve the efficiency and specificity of your multiplex reactions.
Can I perform TA cloning after amplifying an RNA target with the SuperScript IV One-Step RT-PCR System?

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.

What is a two-phase, hot-start activation mechanism?

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.
Can SuperScript IV One-Step RT-PCR Sytem be used with RNA targets with structural complexities?

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.
I am having difficulties performing one-step RT-PCR with long targets. What are your recommendations?

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. Consider optimizing the 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).

For RT-PCR targets up to 13 kb, we recommend SuperScript IV UniPrime One-Step RT-PCR System (Cat. No. 12596025).
Can I use my old one-step RT-PCR cycling protocol with the SuperScript IV One-Step RT-PCR System?

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).
When using the SuperScript IV One-Step RT-PCR System, how do I determine the appropriate annealing temperature for my primers?

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.

For improved performance and simplified workflow, upgrade to the SuperScript IV UniPrime One-Step RT-PCR System (Cat. No. 12596025) which allows universal primer annealing at 60 degrees C for most primer pairs.

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