Invitrogen™ SuperScript™ IV First-Strand Synthesis System

Beschrijving

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