Premix Ex Taq DNA Polymerase (Perfect Real Time) is a 2X RT-PCR kit specifically designed for fast and sensitive real-time PCR via either intercalating green dye Real-Time PCR (qPCR) or probe-based qPCR assays. TB Green dye and probes for probe/5" nuclease-based assays are not included in this RT-PCR kit. The Premix Ex Taq DNA Polymerase (Perfect Real Time) kit consists of our high-fidelity and high-performance Takara Ex Taq Hot Start polymerase and a real-time buffer that ensures superior specificity and increased amplification efficiency during RT-PCR (qPCR). Antibody-mediated hot-start technology prevents nonspecific amplification due to mispriming and/or the formation of primer dimers during room-temperature reaction assembly. Once the Taq antibody-polymerase complex is denatured during the first cycling step, the Premix Ex Taq polymerase can initiate DNA synthesis.
Premix Ex Taq DNA Polymerase (Perfect Real Time) is a 2X RT-PCR kit specifically designed for fast and sensitive real-time PCR via either intercalating green dye real-time PCR (qPCR) or probe-based qPCR assays. TB Green dye and probes for probe/5" nuclease-based assays are not included in this RT-PCR kit. The Premix Ex Taq DNa Polymerase (Perfect RealTime) kit consists of our high-fidelity and high-performance Takara Ex Taq Hot Start polymerase and a real-time buffer that ensures superior specificity and increased amplification efficiency during RT-PCR (qPCR). Antibody-mediated hot-start technology prevents nonspecific amplification due to mispriming and/or the formation of primer dimers during room-temperature reaction assembly. Once the Taq antibody-polymerase complex is denatured during the first cycling step, the Premix Ex Taq polymerase can initiate DNA synthesis.
Two tubes of ROX Reference Dyes are supplied as separate components of this RT-PCR kit. ROX Dye is a convenient internal reference standard that may be used to normalize signal variances that occur as a consequence of non-PCR-related fluorescence fluctuations across different wells or over time.
Premix Ex Taq DNA Polymerase (Perfect Real Time) provides superior specificity, performance, and amplification yield during RT-PCR on all major real-time instruments.
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利用NheI和HindIII双酶切插入目的片段，为什么双酶切有目的片段和切开的质粒，测序却没有？
并且测序后在NheI位点后的序列变成了-GGCTAGGTAC-Kpn位点（CGTTTAAACTTAAGCTTG消失。之前和之后的序列都相符），之间的怎么都没了？
请教高人，这样的问题怎么解决？
多谢
你所说的“测序酶”在一般情况下就是“聚合酶”。
因为目前的的测序方法主要就是借助聚合反应。
当然，有些测序方法（比如曾经的SOLiD系统）是利用连接反应，那么它用的“测序酶”肯定是连接酶。
估计，也就是这个原因有人把它。
两个基因大小分别230kb和450kb，酶切后，450kb的有目的条带，但很弱，230kb的质粒条带亮，其下方有一很微弱条带，用的酶分别是：Ncol和Spel体系是（Takara，两个酶切体系不同，用的官网推荐体系）：
NcoI1μl
Spel1μl
10×KBuffer2μl
0.1%BSA2μl
DNA3ul
灭菌水upto20μl37℃4h电泳1h
期待着高手们的指点，谢谢。
【之前构建的4个重组质粒里也有两个出现这样的问题（aagtcc变成agtcc），但是酶切能切出插入的片段】
目前问测序公司得到的答复是样品可能不是单克隆，测序结果是不准确的，建议重新挑取单克隆测序。
不知道还有没有别的方法？
●应用酶水解多肽不会破坏氨基酸，也不会发生消旋化。水解的产物为较小的肽段。向左转|向右转
