• Oligo(dT)20 (50 µM), 50 µL
• Random hexamers (50 ng/µL), 250 µL
• 5X RT buffer, 1 mL
• 0.1 M DTT, 250 µL
• 10 mM dNTP mix, 250 µL
• SuperScript IV RT (10,000 units total at 200 U/µL), 50 µL
• Ribonuclease Inhibitor (40 U/µL), 100 µL
• E. coli RNase H (2 U/µL), 50 µL
• DEPC-treated water, 1.2 mL
• Total HeLa RNA (10 ng/µL), 20 µL
• Sense Control Primer (10 µM), 25 µL
• Antisense Control Primer (10 µM), 25 µL
• ezDNase Enzyme, 50 µL
• ezDNase Buffer (10X), 100 µL
• Nuclease-free water, 1.25 mL

All components stored at -5 to -30°C.

描述

The SuperScript IV First-Strand Synthesis System with ezDNase Enzyme for RT-PCR is optimized for synthesis of first-strand cDNA from purified poly(A)+ or total RNA. The kit includes SuperScript IV Reverse Transcriptase (RT), a proprietary MMLV mutant that produces robust and reliable cDNA synthesis in RT reactions, and ezDNase 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. This system is also available without ezDNase.

The SuperScript IV First-Strand Synthesis System contains all components needed for RT reactions, plus an additional control gene and primers, and provides the flexibility to customize the RT set-up to fit the needs of your application. For even better performance, ezDNase for genomic DNA removal is included. The extremely simplified genomic DNA removal step 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.

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 15 min including a simplified genomic DNA removal step
• Significantly better processivity compared to SuperScript III RT

Enzyme source
Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability and half-life, processivity, 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.

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