产品描述
 

Hifair^® Ⅱ 1st Strand cDNA Synthesis SuperMix为即用型预混液，包含Hifair^® Ⅱ Reverse Transcriptase，RNase Inhibitor，dNTP，Random primers/Oligo dT Primer mix和优化的缓冲体系，只需再加入模板RNA和RNase-free H₂O即可进行反应。Hifair^® Ⅱ Reverse Transcriptase是在Hieff^® M-MLV (RNase H^–) Reverse Transcriptase基础上通过基因工程技术得到新的逆转录酶，与Hieff^® M-MLV (RNase H^–) Reverse Transcriptase相比，其热稳定性大幅度提高，可耐受高达50℃的反应温度，适合具有复杂二级结构的RNA模板的逆转录。同时，该酶增强了与模板的亲和力，适合少量模板以及低拷贝基因的逆转录。

产品组分
 

产品应用

适用于RT-qPCR实验。

运输和保存方法

冰袋运输。-20℃保存，有效期18个月。

第一链cDNA合成操作步骤

【注】：

1. 20 μL逆转录反应体系建议Total RNA的投入量不超过1 μg。如果目的基因的表达丰度低，最多投入5 μg Total RNA；

2. 逆转录温度：推荐使用42℃。对于高GC含量模板或者复杂模板，可将逆转录温度提高到50℃。

※ 逆转录产物可立即用于后续qPCR反应，也可-20℃短期保存，若需长期保存，建议分装后，于-80℃保存，避免反复冻融。

注意事项

1）所有操作均应在冰上进行，且操作过程应避免RNase污染；

2）为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
3）本产品仅作科研用途！

相关产品
 

HB210719

Q：逆转录反应中的引物如何选择？

A：根据实验目的不同，可按下列建议选择（选择指南可见下表）：

a）对于全长第一链 cDNA 合成，且模板为真核生物来源，推荐选择Oligo (dT)引物。

b）当目标区域具有复杂二级结构或模板为原核生物来源，推荐选择 Random Primers 引物。

c）基因特异性引物（GSP）是与模板序列互补的引物，适用于目的序列已知的情况。

d）若逆转录后续为 qPCR 实验，可将 Oligo (dT)与Random Primers 混合使用。

A：有 A 尾的 lncRNA 用预混液和 KIT （ 11123/11141/11121/11139） 都可以， circRNA、miRNA 和无 A 尾的 lncRNA 用  KIT（11121/11139）。microRNA 需要特殊的茎环引物，需特别针对的逆转录试剂盒。

Q：能否用来做 miRNA/circRNA/lncRNA 的逆转录？

A：RNA 的物种与逆转录没有很大关系，RNA 质量与逆转录有关。所以，得到 RNA 后， 逆转录试剂盒都可以用，没有特别针对的。

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Q：克隆逆转和定量逆转有什么区别？克隆逆转产物和定量逆转产物可以相互吗？

A：a）目的不同：克隆逆转后的 cDNA 后续用于基因克隆，后续实验以普通 PCR 为主； 定量逆转后的 cDNA 后续用于基因定量，后续实验以qPCR 为主。

b）逆转录过程不同：克隆逆转使用 Oligo dT，保证合成的长度。随机引物使用较少； 定量逆转使用 Oligo dT 或随机引物，或两者混合使用。

c）克隆逆转产物可用于 qPCR，但定量逆转产物不推荐用于普通 PCR，定量逆转试剂中含 Oligo (dT)与 Random Primers 混合引物，产物长度较短，可做短片段 PCR，过长的不适合。

Q：逆转录试剂盒可以逆转真菌（或其他物种）RNA 吗？有没有专门针对真菌（或其他物种）的RNA 逆转录的试剂盒呢？