产品说明书
FAQ
COA
已发表文献
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
|
编号 |
组分 |
产品编号/规格11120ES60 (100 T) |
|
11120-A |
RNase-free H2O |
2×1 mL |
|
11120-B |
2×Hifair® Ⅱ SuperMix |
1 mL |
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
|
逆转录反应体系 |
逆转录程序 |
||
|
组分 |
使用量 |
温度 |
时间 |
|
RNase free H2O |
To 20 μL |
25℃ |
5 min |
|
2×Hifair® Ⅱ SuperMix |
10 μL |
42℃ |
30 min |
|
Total RNA |
1 ng -5 μg |
85℃ |
5 min |
|
or mRNA |
1 ng-500 ng |
||
【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！
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11119ES60 |
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Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) |
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Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，Low Rox) |
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|
Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，High Rox) |
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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。
|
逆转录引物选择指南 |
||||
|
|
特征 |
优点 |
缺点 |
结合方式 |
|
Oligo (dT) |
1）12-20 个T； 2）与真核生物 mRNA 的3 ’ Poly A 尾配对。 |
可 合 成 全长 cDNA。 |
1 ） 仅扩增有 polyA尾 的mRNA； 2 ） 对模板质 量 要 求高。 |
|
|
Random Primers |
1）6-9 个碱基； 2）可随机识别模板并结合 |
适 合 复 杂结 构 和 微量模板。 |
特异性低， 小片段多。 |
|
|
基因特异性性引物 （GSP） |
识别特定模板序列。 |
特异性强， 灵敏度高。 |
仅 合 成 特定的序列。 |
|
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？
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？
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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