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內(nèi)容及儲存
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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
RNaseOUT? (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
All components stored at -5 to -30°C.
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描述
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The SuperScript? IV First-Strand Synthesis System 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. 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 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. The SuperScript? IV synthesis system 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 10 min? Significantly better processivity compared to SuperScript? III RTEnzyme sourcePurified 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 testingEndodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product.Unit definitionOne 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 conditions50 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.