| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| F665646-5ml |
5ml |
期货  |
| |
| F665646-40ml |
40ml |
期货 |
|
| 别名 | 2×Flash Hot Start MasterMix(染料) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | 2×Flash Hot Start MasterMix (Dye) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 储存温度 | -20°C储存,避免反复冻融 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品简介 本品是由一种新型高效的DNA聚合酶、PCR Buffer、Mg2+、dNTPs以及PCR稳定 剂和增强剂组成的预混体系，浓度为2×。内含新型高效热启动酶，能够在低温条件下 有效抑制引物的非特异性退火及引物二聚体引起的非特异性扩增。本品具有极高的扩 增速度与稳定性，延伸速度可达5-15 sec/kb，适用于快速PCR反应，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。主要适用于快速PCR反应和对高保真性有要求的基因克隆等 实验。
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：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：如扩增样本为菌液需增加“预变性95℃ 5min”步骤。 优化参数设定 1． 模板DNA量设定： 模板过量可能导致非特异性扩增或smear。50 μl PCR反应体系中模板DNA推荐使 用量如下： -人基因组DNA 5 ng-500 ng -大肠杆菌基因组DNA 50 pg-100 ng -质粒DNA 10 pg-1 ng 2． 引物浓度设定： 引物浓度可设为0.1μM-1.0 μM 之间。引物浓度过低时可能导致扩增产物少。引物 浓度过高会抑制特异性扩增，可能导致非特异性扩增。 3． 退火温度设定： 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 时可适当降低退火温度；发生非特异性反应时可适当提高退火温度。对于复杂模 板，需要调节退火温度实现高效扩增。 4． 延伸时间设定： 延伸时间应根据所扩增片段大小设定。以下为推荐的延伸时间： 质粒等简单模板：5-15 s/kb； 常规基因组、cDNA模板：10-15 s/kb； 复杂模板、粗提模板：20-30 s/kb； （延伸时间不宜过短应至少在5 s/kb以上，也不宜超过30 s/kb）。
5．
循环数设定：
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。
Products Introduction This product is a premixed system consisting of a new type of highly efficient DNA polymerase, PCR Buffer, Mg2+, dNTPs, and PCR stabilizers and enhancers at a concentration of 2 ×. It contains a new type of high efficient hot starter enzyme, which can effectively inhibit the non-specific annealing of primers and non-specific amplification caused by primer dimer under low temperature. The product has a very high amplification speed and stability, the extension speed can be up to 5-15 sec/kb, suitable for rapid PCR reaction, the original MasterMix formula makes the whole reaction system very stable, more than 98% of the PCR amplification can be successful at once, at the same time, complex templates can also be amplified effectively, and can minimize the human error and contamination. The product has added dye (blue), and can be directly detected by electrophoresis after the reaction. Most of the amplified PCR products have an "A" base at the 3′ end, so they can be used directly for T/A cloning. It is mainly used for rapid PCR reactions and gene cloning where high fidelity is required. Quality control Tested to be free of exogenous nuclease activity; PCR method detects no host residual DNA; can efficiently amplify a wide range of base Single-copy genes in the genome. Usage The following is an example of a PCR reaction system and reaction conditions using human genomic DNA as a template, and the actual operation should be based on the template. Plates, primer structures and target fragment sizes were improved and optimized accordingly. PCR reaction system
Note: Please use the final concentration of 0.1-1.0 μM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the primer can be increased. concentration; when a non-specific reaction occurs, the primer concentration can be reduced, thus optimizing the reaction system. PCR reaction conditions
Note: If the amplified sample is bacterial liquid, add "pre-denaturation 95℃ for 5min" step. Optimization of parameter settings 1. Template DNA amount setting: Excessive amounts of template may result in non-specific amplification or smear. The recommended amount of template DNA in a 50 μl PCR reaction system is as follows: -Human genomic DNA 5 ng-500 ng -Escherichia coli genomic DNA 50 pg-100 ng -plasmid DNA 10 pg-1 ng 2. Primer concentration setting: The primer concentration can be set between 0.1 μM and 1.0 μM. Too low a primer concentration may result in low amplification products. Too high a primer concentration will inhibit specific amplification and may result in non-specific amplification. 3. Annealing temperature setting: In general, the annealing temperature in the experiment is 5℃ lower than the melting temperature of the amplification primer, Tm, which is not able to get the ideal amplification efficiency. The annealing temperature can be lowered appropriately in the case of non-specific reactions; the annealing temperature can be increased appropriately in the case of non-specific reactions. For complex templates, the annealing temperature needs to be adjusted to achieve efficient amplification. 4. Extension time setting: The extension time should be set according to the size of the amplified fragments. The following are recommended extension times: Simple templates such as plasmids: 5-15 s/kb; Conventional genome, cDNA templates: 10-15 s/kb; Complex templates, crude extraction templates: 20-30 s/kb; (The extension time should not be too short it should be at least 5 s/kb and not more than 30 s/kb). 5. Cycle number setting: The number of cycles can be set according to the downstream application of the amplified product. If the number of cycles is too low, the amount of amplification will be insufficient; if the number of cycles is too high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield. |
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