SuperScript IV CellsDirect cDNA 合成试剂盒包括 SuperScript IV 反转录酶。该酶的特点包括: •更低的 RNase H 活性 • 更高的热稳定性 • 在第一链合成反应中产生高产量 cDNA,以便提高灵敏度并增强罕见转录本的检测 • 对各种 RT 抑制剂具有优异的耐受性
SuperScript IV 反转录酶是所有 RT-PCR 和 qRT-PCR 应用的极佳选择。这是一种专有 MMLV 突变体,在 RT 反应中具有出色的耐用性和可靠性。与 SuperScript III 反转录酶相比,在抑制剂耐受性、持续合成能力和反应速度等方面显著改善,同时保留了前代酶的所有优点,包括较高的热稳定性、高效全长 cDNA 合成以及较低的 RNase 酶活性。
工作原理 在传统的 RT-PCR 中,首先从细胞中分离出 RNA,这个程序非常耗时,会导致材料损失。使用 SuperScript IV CellsDirect cDNA 合成试剂盒,可在单管中将细胞裂解,并以尽可能少的操作量从裂解物中获得 cDNA,且无样品损失。DNase I 处理是可选步骤,可用于在第一链合成前消除基因组 DNA。该试剂盒已针对小细胞样品(从 10,000 个细胞到单个细胞不等)进行了优化。
规格
最终产品类型第一链 cDNA
产品规格试剂盒
环保功能危害少、废弃物少
反应次数500 次反应
最佳反应温度50°C
数量500 reactions
反应形式预混组分
试剂类型反转录
逆转录酶SuperScript IV
核糖核酸酶 H 活性减少
运输条件干冰
原始材料细胞
技术反转录
适用于(应用)RT-PCR, 实时 PCR (qPCR)
高 GC PCR 扩增效果高
反应速度10 min。
Unit SizeEach
内容与储存
• SuperScript IV CellsDirect 裂解液 (3 x 9 mL) • SuperScript IV CellsDirect 终止液 (2 x 1.25 mL) • 裂解增强剂 (1 x 250 µL) • DNase I (1 x 500 µL) • SuperScript IV RT 预混液 (4 x 1 mL) • SuperScript IV 无 RT 对照品 (4 x 1 mL)
储存在 –20°C 下。
常见问题解答 (FAQ)
What are the RT primers included in the SuperScript IV CellsDirect cDNA Synthesis Kit (Cat. No. 11750150, 11750350)?
The RT master mix contains both random and oligo dT primers.
In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will active ezDNase affect RNA or the RT reaction?
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°C). The additional inactivation step is therefore not required in RT-qPCR applications.
Which SuperScript IV RT format do you recommend for real-time PCR applications?
For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). 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.
Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?
The only change is that the incubation time for the reverse transcription reaction has been reduced from 50 minutes to 10 minutes. All the other parameters and steps are the same.
Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?
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).