计算溶液所需的质量、体积或浓度。
,适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
本品是由Taq Plus DNA Polymerase、Mg2+、dNTPs以及PCR稳定剂和增强剂组 成的预混体系,浓度为2×。与Taq DNA Polymerase相比,Taq Plus DNA Polymerase 扩增效率更高,保真性更优秀。独创的MasterMix配方使整个反应体系非常稳定,超过 98%的PCR扩增能一次成功,同时复杂模板也能得到有效扩增,并可最大限度地减少 人为误差和污染。本产品已加入染料(蓝色),反应结束后可直接进行电泳检测。扩 增得到的大部分PCR产物3′端附有一个“A”碱基,因此可直接用于T/A克隆。主要适用于 常规PCR反应和对高保真性有要求的基因克隆等实验。
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质量控制
经检验无外源核酸酶活性;PCR方法检测无宿主残余DNA;能有效地扩增多种基 因组中的单拷贝基因。
使用方法
以下举例为以人基因组DNA为模板,扩增1 kb的片段的PCR反应体系和反应条件,实 际操作中应根据模板、引物结构和目的片段大小不同进行相应的改进和优化。
1. PCR反应体系
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注意:引物浓度请以终浓度0.1-1.0 μM作为设定范围的参考。扩增效率不高的情况下,可提高 引物的浓度;发生非特异性反应时,可降低引物浓度,由此优化反应体系。
2. PCR反应条件
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注意:
1)一般实验中退火温度比扩增引物的熔解温度Tm低5℃,无法得到理想的扩增效率时,适当降 低退火温度;发生非特异性反应时,提高退火温度,由此优化反应条件。
2)延伸时间应根据所扩增片段大小设定,Taq Plus DNA Polymerase的扩增效率为2 kb/min。
3)可根据扩增产物的下游应用设定循环数。如果循环次数太少,扩增量不足;如果循环次数太
多,错配机率会增加,非特异性背景严重。所以在保证产物得率的前提下应尽量减少循环次数。
This product is a premixed system composed of Taq Plus DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. Compared with Taq DNA Polymerase, Taq Plus DNA Polymerase has higher amplification efficiency and better fidelity. The unique MasterMix formula makes the entire reaction system very stable, with over 98% of PCR amplification successful at once. At the same time, complex templates can also be effectively amplified, and human error and contamination can be minimized to the greatest extent. This product has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. Most PCR products obtained from amplification have an "A" base attached to the 3 'end, making them suitable for direct use in T/A cloning. Mainly suitable for routine PCR reactions and gene cloning experiments that require high fidelity.
Quality control
After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes.
Usage
The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.
1. PCR reaction system
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Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.
2. PCR reaction conditions
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Attention:
1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.
2) The extension time should be set according to the size of the amplified fragment, and the amplification efficiency of Taq Plus DNA Polymerase is 2 kb/min.
3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible.