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Invitrogen™ SuperScript™ IV First-Strand Synthesis System
Description
The SuperScript IV First-Strand Synthesis System for RT-PCR, available with or without ezDNase, is optimized for synthesis of first-strand cDNA from purified poly(A)+ or total RNA. The system includes SuperScript IV Reverse Transcriptase (RT), a proprietary MMLV mutant that produces robust and reliable cDNA synthesis in RT reactions. The SuperScript IV First-Strand Synthesis System with ezDNase has the further ability to remove potential genomic DNA contamination.
The SuperScript IV synthesis system is significantly improved over the SuperScript III-based system in inhibitor resistance, processivity, and reaction speed, while retaining all the benefits of the previous system, including increased thermostability, highly efficient full-length cDNA synthesis, and reduced RNase activity. The SuperScript IV system is designed to provide reliable, consistent, and fast cDNA synthesis in the presence of inhibitors found in a wide variety of samples that cause other currently available RTs to perform inefficiently.
The extremely simplified genomic DNA removal step of the SuperScript IV First-Strand Synthesis System with ezDNase dramatically reduces the time of the entire reverse transcription protocol and reduces possible variation in gene expression due to RNA loss or damage during conventional DNase treatment. The SuperScript IV synthesis system with ezDNase is the top choice for performance and flexibility for RT-PCR applications. We recommend it for all RT-PCR applications, especially when reproducibility and reliability is the primary concern and when inhibitors in the RNA sample may interfere with cDNA synthesis, leading to biases in gene expression studies.
Both SuperScript IV First-Strand Synthesis systems contain all components needed for RT reactions, plus an additional control gene and primers, and provide the flexibility to customize the RT set-up to fit the needs of your application.
Features of the SuperScript IV First-Strand Synthesis System include:
• Significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis
• Robust and specific cDNA synthesis in a wide range of sample types
• A faster reverse transcriptase reaction that reduces incubation time from >50 min to 10 min
• Significantly better processivity compared to SuperScript III RT
• Simplified genomic DNA removal step (SuperScript IV synthesis system with ezDNase only)
Enzyme source
Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability, half-life, processivity, and inhibitor resistance and to reduce RNase H activity.
Performance and quality testing
Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product
Unit definition
One unit of SuperScript IV RT is the amount of enzyme required to incorporate 1 nmole of deoxyribonucleotide into acid-precipitable material in 10 min at 37°C using poly(A) oligo(dT)12-18 as a template/primer.
Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 4 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.5 mM dTTP, 0.4 MBq/mL [3H]-dTTP, 0.4 mM poly(A) oligo (dT)12-18 and enzyme in 20 μL for 10 min at 37°C
Specifications
Specifications
| Content And Storage | • Oligo(dT)20 , 50 μL (50 μM) |
| Format | Kit |
| GC-Rich PCR Performance | High |
| Reaction Speed | 10 min. |
| Technique | Reverse Transcription |
| Optimal Reaction Temperature | 50°C |
| Quantity | Each |
| Reverse Transcriptase | SuperScript IV |
| Shipping Condition | Dry Ice |
| For Use With (Application) | RT-PCR, Real Time PCR (qPCR) |
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Frequently Asked Questions (FAQs)
Yes. The SuperScript IV First-Strand Synthesis System (Cat. No. 18091050, 18091200) "No Reverse Transcriptase (RT) Control" will often display a level of amplification because of residual genomic DNA in the RNA preps. In general, you do not need to worry if a faint band shows up on a gel, as long as the intensity of the positive control is much higher.
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.
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.
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.
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).
For Research Use Only. Not for use in diagnostic procedures.