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- 详细信息
- 询价记录
- 文献和实验
- 技术资料
- 保存条件:
-20℃保存
- 保质期:
有效期18个月
- 英文名:
Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)
- 库存:
大量
- 供应商:
翌圣生物科技(上海)股份有限公司
- 规格:
10T(11141ES10)/100T(11141ES60)
| 规格: | 10T(11141ES10) | 产品价格: | ¥326.00 |
|---|---|---|---|
| 规格: | 100T(11141ES60) | 产品价格: | ¥1383.00 |
该预混液包含5×gDNA digester Mix和4×Hifair® Ⅲ SuperMix plus。5×gDNA digester Mix可去除RNA模板中残留的基因组DNA污染,保证后续结果更加可靠。4×Hifair® Ⅲ SuperMix plus 含有逆转录反应所需的所有组分(Buffer,dNTP,Hifair® Ⅲ Reverse Transcriptase,RNase inhibitor,Random primers/ Oligo (dT)18 primer mix),只需加入RNA模板和 RNase-free H2O即可进行逆转录反应,并同时终止gDNA digester的作用,保证cDNA的完整性。
该产品适用于两步法RT-qPCR检测,针对qPCR进行特别优化,比例优化的Random primers/ Oligo (dT)18 primer mix,使cDNA合成可从RNA转录本的各个区域起始,并具有相同的逆转录效率,保证了qPCR结果的真实性和可重复性。逆转录产物兼容SYBR® Green和探针法qPCR,可以根据实验目的,选择Hieff UNICON® qPCR SYBR® Green Master Mix,Hieff® qPCR SYBR® Green Master Mix或Hieff® qPCR TaqMan Probe Master Mix等试剂配合使用,进行高性能的基因表达分析。

产品组分
|
组分编号 |
组分名称 |
产品编号/规格 | |
|
11141ES10 (10 T) |
11141ES60 (100 T) | ||
|
11141-A |
RNase-free H2O |
1 mL |
2×1 mL |
|
11141-B |
5×gDNA digester Mix |
30 μL |
320 μL |
|
11141-C |
4×Hifair® Ⅲ SuperMix plus |
50 μL |
500 μL |
【注】:4×Hifair® Ⅲ SuperMix plus含有gDNA digester抑制剂。
产品应用
适用于两步法RT-qPCR。
运输和保存方法
冰袋运输。-20℃保存。有效期18个月。
注意事项
1)5×gDNA digester Mix和4×Hifair® Ⅲ SuperMix plus含有高浓度的甘油,使用前请短暂离心收集到反应管底部,并用移液枪轻轻吸打充分混匀后,准确吸取;
2)如果加入RNA模板体积较大 (如超过2 μL),请确保RNA是溶于水中而不是TE中,因为TE会对gDNA去除以及逆转录反应产生抑制;
3)可以不经过基因组去除步骤,直接用4×Hifair® Ⅲ SuperMix plus进行逆转录,这样所得到的结果会与使用Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (Cat No.11137ES) 效果一致。但是请勿将gDNA digester与11137ES中的Hifair® Ⅲ SuperMix配套使用,因其不含终止gDNA digester反应的成分,会影响反转录和后续的qPCR实验;
4)反应液的配制应在冰上操作完成,操作过程应避免RNase污染;
5)为了您的安全和健康,请穿实验服并佩戴一次性手套操作;
6)本产品仅作科研用途!
第一链cDNA合成操作步骤
1. 残留基因组DNA去除
在RNase free离心管中配制如下混合液,用移液器轻轻吹打混匀。42℃孵育2 min。
| 组分 | 使用量 |
| RNase-free H2O | To 15 μL |
| 5×gDNA digester Mix | 3 μL |
| Total RNA or mRNA | 10 pg -5 μg* 10 pg-500 ng* |
2. 逆转录反应体系配制(20 μL体系)
在第1步的反应管中直接加入4×Hifair® Ⅲ SuperMix plus,用移液器轻轻吹打混匀。
| 组分 | 使用量 |
| 第1步的反应液 | 15 μL |
| 4×Hifair® Ⅲ SuperMix plus | 5 μL |
| 温度 | 时间 |
| 25℃ | 5 min |
| 55℃ | 15 min |
| 85℃ | 5 min |
逆转录快速程序设置
| 温度 | 时间 |
| 55℃ | 15 min |
| 85℃ | 5 sec |
※ 逆转录产物可立即用于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。
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- 作者
- 内容
- 询问日期
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RTase 两大类:AMV RTase 的 RNase H 活性强,合成长度短,合成量低,热稳定性好(42~55℃);MMLV RTase 的 RNase H 活性弱,合成长度长,合成量高,热稳定性差(37~42℃)。由于 RNase H 酶具有降解 RNA 模板的功能,所以在逆转录时应该优先选择 RNase H 活性弱的 MMLV ,而且后期经过基因工程改造,MMLV 热稳定性已达到质的飞跃。以 Yeasen 的 Hifair® Ⅲ 逆转录酶(11141)为例,该酶经基因工程改造,删除了 RNase
录不一样。现在常见的microRNA反转录引物有2种,一个是step-loop结构;另外一种是3末端加polyA后,再用oligo(dT)逆转录。BioTeke有microRNA提取和2-step real-time PCR Kit。 3. cDNA的合成(RT)电泳,以及cDNA第二链的合成问题? 2-5uLcDNA产物电泳有模糊条带出现。第二链合成也有专门的试剂盒。BioTeke现在没有此类产品。 4. Power Mix扩增不成功因素? 产品
一种用于肝素粗品质量监测的、检测原料药中反刍动物DNA的多重RT-PCR分析方法的开发
离心5s。 e. 在检测每一批待测样品的同时,必须设置珠子空白对照(珠子+无DNA的水)分析。 3.2. 步骤 a. 移取25 μL先前准备好的预混液(珠子和水)至SmartCycler PCR反应管。 b. 向每一根对应的PCR反应管中加入1 μL样品。 c. 盖上管盖,在SmartCycler离心机中离心5 s。 d. 将PCR反应管放入SmartCycler区块





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