计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
本品为Pfu 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×。Pfu DNA Polymerase具有5′-3′ DNA聚合酶活性和3′-5′外切酶活性，因此在DNA扩增过程中具备纠错能力。与Taq DNA Polymerase相比具有高保真性（Taq酶的6-8倍）和更好的热稳定性。预配好的PCR混合液使操作更加简单快捷，可最大限度地减少人为误差和污染。独创的MasterMix配方使整个反应体系非常稳定，重复性好。本产品已加入染料（蓝色），反应结束后可直接进行电泳检测。本产品所含的Pfu DNA聚合酶具有错配率低，耐高温的特点，适用于基因克隆、基因定点突变、SNP和末端补平等反应。
Notes: 2×Pfu MasterMix contains Pfu DNA Polymerase, 3mM MgCl2 and 400µM each dNTP. |
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；2-8℃存放三个月，活性无明显改变。
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
|
注意：用Pfu酶扩增时，引物的纯度要求较高，引物长度大于18个碱基，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
|
注意：
1）Pfu酶的热稳定性比Taq酶好，对于GC含量很高的模板，变性温度可以提高到98℃，对Pfu酶 的活性无影响。
2）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。
3）Pfu酶具有3′-5′外切酶活性，所以Pfu酶扩增时延伸速度远比Taq酶低，延伸时间根据所扩增 片段大小设定，本产品的扩增延伸速率为1 kb/min。
4）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。
5）本产品具有3′-5′外切核酸酶活性，PCR产物为平端不能直接用于T/A克隆，如需进行T/A克隆
则需要在其末端添加“A”或用平末端载体进行克隆。
This product is a premixed system composed of Pfu DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers, with a concentration of 2 ×. Pfu DNA Polymerase exhibits 5 ′ -3 ′ DNA polymerase activity and 3 ′ -5 ′ exonuclease activity, thus possessing error correction ability during DNA amplification. Compared with Taq DNA Polymerase, it has high fidelity (6-8 times that of Taq enzyme) and better thermal stability. The pre prepared PCR mixture makes the operation simpler and faster, minimizing human error and contamination to the greatest extent possible. The original MasterMix formula makes the entire reaction system very stable and has good repeatability. This product has been added with a dye (blue), and can be directly subjected to electrophoresis detection after the reaction is completed. The Pfu DNA polymerase contained in this product has the characteristics of low mismatch rate and high temperature resistance, making it suitable for gene cloning, gene directed mutagenesis, SNP and end effector complement reactions.
Notes: 2×Pfu MasterMix contains Pfu DNA Polymerase, 3mM MgCl2 and 400µM each dNTP. |
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; Storage at 2-8 ℃ for three months showed no significant change in activity.
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
|
Attention: When amplifying with Pfu enzyme, the purity of the primers is required to be high, and the length of the primers is greater than 18 bases. The primer concentration should be based on the final concentration of 0.1-1.0 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
|
Attention:
1) The thermal stability of Pfu enzyme is better than that of Taq enzyme. For templates with high GC content, the denaturation temperature can be increased to 98 ℃ without affecting the activity of Pfu enzyme.
2) 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.
3) Pfu enzyme has 3 '-5' exonuclease activity, so the extension rate of Pfu enzyme during amplification is much lower than that of Taq enzyme. The extension time is set according to the size of the amplified fragment, and the amplification extension rate of this product is 1 kb/min.
4) 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.
5) This product has 3 '-5' exonuclease activity. The PCR product is a flat end and cannot be directly used for T/A cloning. If T/A cloning is required, "A" needs to be added at its end or cloned using a flat end vector.
规格
别名
2xPfu MasterMix（染料）
规格或纯度
5 ml
英文名称
2xPfu MasterMix（Dye）
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
产品线
biological_products
名称和识别符
分子类型
小分子
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
Copyright © 2023至今 上海阿拉丁生化科技股份有限公司 保留所有权利。
危险品化学品经营许可证（带存储）