The TotalPrep™-96 RNA Amplification Kit is a complete system for generating biotinylated, amplified RNA for hybridization with Illumina Sentrix® arrays. The kit includes sufficient reagents for 96 reactions. Features of the TotalPrep™-96 RNA Amplification Kit: • Perform amplification from just 50 ng of input RNA • Developed for expression profiling using the Sentrix® BeadChips • Bio-16-UTP now included in optimized NTP mix, reducing set-up time and maximizing labeling Using the TotalPrep™-96 RNA Amplification Kit The TotalPrep™-96 RNA Amplification Kit incorporates extensive improvements to the in vitro transcription reaction (IVT) over the previous TotalPrep™ Kit. The IVT reaction has been optimized for the most effective biotin labeling. An NTP mix containing biotinylated UTP has been added to the kit, allowing ease of use and optimal labeling. These modifications to the kit have significantly increased sensitivity, providing increased Percent Present calls and single-round amplification with just 50 ng of input RNA. The kit is based on the RNA amplification protocol developed in the laboratory of James Eberwine (Van Gelder et al. 1990). The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using ArrayScript™, a reverse transcriptase (RT) engineered to produce higher yields of first strand cDNA than wild type enzymes. ArrayScript™ catalyzes the synthesis of virtually full-length cDNA, which is the best way to ensure production of reproducible microarray samples. The cDNA then undergoes second strand synthesis and magnetic-based cleanup to become a template for in vitro transcription with T7 RNA Polymerase. To maximize cRNA yield, MEGAscript® in vitro transcription (IVT) technology, along with biotin UTP (provided in the kit), is used to generate hundreds to thousands of biotinylated, antisense RNA copies of each mRNA in a sample. Accessory product A 96-well magnetic-ring stand is required to use this kit. The 96-well Magnetic-Ring Stand (Cat. No. AM10050) is recommended and designed for paramagnetic bead precipitation from standard 96-well U-bottom microplates. The Magnetic-Ring Stand pellets the magnetic beads in a circle shape, enabling easy removal of supernatant by a robot or multichannel pipettors. 规格 包括标签或染料 是 标记方法 直接标记 产品线 Ambion™、Illumina™ TotalPrep™ 数量 96 Reactions 逆转录酶 ArrayScript™ 样品类型 poly(A+) RNA、总 RNA 系统类型 Illumina Sentrix™ 产品类型 Kit, RNA Amplification 内容与储存 • 110 µl T7 Oligo(dT) 引物 (-20°C) • 110 µl ArrayScript™ 逆转录酶 (-20°C) • 220 µL 10X 第一链缓冲液 (-20°C) • 880 µL dNTP 混合物 (-20°C) • 110 µl RNase 抑制剂 (-20°C) • 110 µL RNase H (-20°C) • 1.1 mL 10X 第二链缓冲液 (-20°C) • 220 µL DNA 聚合酶 (-20°C) • 275 µL T7 酶混合物 (-20°C) • 275 µL 生物素-NTP 混合物 (-20°C) • 10 µL 对照 RNA (-20°C) • 20 mL cDNAPure (4°C) • 1.1 mL RNA 结合珠 (4°C) • 40 mL cDNA 洗涤缓冲液浓缩液（室温） • 5.5 mL cRNA 结合缓冲液浓缩液（室温） • 450 µL 微珠重悬溶液（室温） • 25 mL cRNA 洗涤液浓缩液（室温） • 10 mL cRNA 洗脱缓冲液（室温） • 8 个 U 形底板（室温） • 8 个 PCR 板（室温） • 20 mL 无核酸酶水（任意温度）