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2.5×TFS Master Mixture
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| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| T666123-1mL |
1ml |
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基本描述
| 别名 | 2.5×TFS 母液 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 规格或纯度 | 1 mL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 英文名称 | 2.5×TFS Master Mixture | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 2.5×TFS Master Mixture是一款适用于各种类型多重PCR的预混体系，浓度为 2.5×，包含DNA聚合酶、PCR Buffer、dNTPs、Mg2+以及稳定剂和增强剂等成分，操 作简便快速。 2.5×TFS Master Mixture包含的DNA聚合酶是一种经基因工程改造的重组酶，具有 5’→3’DNA聚合酶活性，无5’→3’外切酶活性；DNA聚合酶经过新型抗体修饰，为抗体 修饰热启动酶，能够有效减少在常温条件下由引物和模板非特异性结合或引物二聚体 而产生的非特异性扩增，同时具有激活时间短、扩增能力强、灵敏度高、稳定性好等 优良特点。独特的PCR缓冲体系与热启动酶的组合，显著提高了PCR的扩增效率，灵 敏度更高，抑制物耐受性更强。 该产品应用范围广，不仅适用于普通和染料法实时荧光定量PCR，更适用于法医 多重STR扩增反应，可用于法医学分析、亲权鉴定以及科研等人类遗传鉴定方面。 注意事项 1. 使用前请在本品完全融化后上下颠倒轻轻混匀，并经短暂离心后使用。 2. 避免反复冻融本品，反复冻融可能使产品性能下降。本品可置于-20℃长期保存。 使用方法 以下举例为STR检测反应体系和反应条件，实际操作中应根据具体用途、模板、引物 结构、目的片段大小和扩增效果不同进行相应的改进和优化。 1.PCR反应体系 提取DNA扩增反应体系：
注意： 1） 引物设计时，应尽量减小各引物的Tm间的差值，差值尽量控制在5℃以内。扩增效率不高的情况 下，可提高引物浓度；发生非特异性扩增时，可降低引物浓度，由此优化反应体系。为达到最优扩增 效果，建议引物混合物使用前涡旋震荡10 s短暂离心后使用。 2） 通常DNA模板的量以0.1 ng-1 ng人基因组DNA为参照，可根据扩增效果调整模板投入量，以确 定最佳的模板使用量。 3） 操作过程中应避免人源基因组污染，推荐实验时设置一组阴性对照（无DNA）。 2. PCR反应条件
注意： 1） 推荐采用两步法PCR反应程序。若因引物Tm值较低或引物间Tm值相差较大等原因，得不到良好 的实验结果时，可尝试使用三步法PCR扩增，退火温度请以55℃-65℃的范围作为设定参考（退火温 度通常比Tm值低5℃），延伸温度设置为72℃。 2） 当得不到良好的扩增效果时，可适当延长退火、延伸时间至120 s-150 s。 3） 当PCR产物检测出现加A不完全时，可适当延长终延伸时间至30-40 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，推荐循环数为28-31 个循环。 5） 血卡直扩可根据实际扩增效果增加72℃裂解步骤以提高扩增效率。 6） 当使用ABI 9700热循环仪时，请在MAX模式下进行扩增。 7） PCR产物可置于2-8℃短期保存，也可置于-20℃长期保存。 Products content
Products Introduction The 2.5×TFS Master Mixture is a premixed system for all types of multiplexed PCRs. It contains DNA polymerase, PCR Buffer, dNTPs, Mg2+, stabilizers and enhancers at a concentration of 2.5×, which makes it easy and fast to use. The DNA polymerase contained in the 2.5×TFS Master Mixture is a genetically engineered recombinant enzyme with 5'→3' DNA polymerase activity and no 5'→3' exonuclease activity; it is a new antibody-modified hot-start enzyme that can effectively reduce non-specific amplification caused by non-specific binding of primer and template or primer dimerization at room temperature. The DNA polymerase is modified by a new type of antibody, which is an antibody-modified hot-start enzyme, and can effectively reduce the non-specific amplification caused by the non-specific binding of primers and templates or primer dimerization at room temperature, and at the same time, it has the excellent features of short activation time, strong amplification ability, high sensitivity, good stability, etc. The unique PCR buffer system and the hot-start enzyme are suitable for the PCR. The unique combination of PCR buffer system and hot starter enzyme significantly improves the PCR amplification efficiency, sensitivity and inhibitor tolerance. The product has a wide range of applications, not only for general and dye-based real-time fluorescence PCR, but also for forensic multiple STR amplification reaction, which can be used in forensic analysis, parentage identification and scientific research and other human genetic identification.
caveat 1. Before use, please mix the product gently by turning it up and down after it is completely melted and centrifuged briefly. 2. Avoid repeated freezing and thawing of the product, which may degrade its performance. This product can be stored at -20℃ for a long time.
Usage The following examples are STR reaction systems and conditions, which should be improved and optimized according to the specific use, template, primer structure, target fragment size and amplification effect. PCR reaction system Extract the DNA amplification reaction system:
Blood Card Direct Expansion Reaction System:
Attention: 1) When designing the primers, the difference between the Tm of each primer should be minimized, and the difference should be controlled within 5℃ as far as possible. If the amplification efficiency is not high, the concentration of primers can be increased; if non-specific amplification occurs, the concentration of primers can be decreased to optimize the reaction system. For optimal amplification, it is recommended that the primer mixture be vortexed for 10 s and centrifuged briefly before use. (2) The amount of DNA template is usually 0.1 ng-1 ng of human genomic DNA as a reference, and the amount of template input can be adjusted according to the amplification effect to determine the optimal amount of template to use. 3) Human genome contamination should be avoided during the operation, and a set of negative control (no DNA) is recommended for the experiment. 2. PCR reaction conditions
Attention: 1) Two-step PCR reaction program is recommended. If you can not get good results due to low Tm value of the primers or large difference in Tm value between primers, you can try to use three-step PCR amplification, the annealing temperature should be set in the range of 55℃-65℃ as a reference (the annealing temperature is usually 5℃ lower than the Tm value), and the extension temperature should be set at 72℃. (2) When good amplification results are not obtained, the annealing and extension time can be appropriately prolonged to 120 s-150 s. The annealing and extension time can be extended to 120 s-150 s. (3) When the PCR product detection appears to be incomplete plus A, the final extension time can be appropriately extended to 30-40 min. 4) The number of cycles can be set according to the downstream application of the amplified product, if the number of cycles is too small, the amplification is insufficient, the recommended number of cycles is 28-31 cycles. 5) Blood card direct amplification can be based on the actual amplification effect to increase the 72 ℃ lysis step to improve the amplification efficiency. 6) When using the ABI 9700 Thermal Cycler, perform amplification in MAX mode. 7) PCR products can be stored at 2-8°C for short-term storage or at -20°C for long-term storage.
Attention: (1) Usually, a primer concentration of 0.2 M can give better results, and 0.1-1.0 μM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; if non-specific reaction occurs, the concentration of primer can be decreased to optimize the reaction system. (2) The final concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe to adjust the concentration. 3) Usually the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference, as templates of different species The number of copies of the target gene contained in them varies, and a gradient dilution of the template can be performed to determine the optimal amount of template to use.
PCR reaction conditions
: (1) The enzyme used in this product is activated by pre-denaturation at 95°C for 30 s. Most of the templates can be deconvoluted well under this condition. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1 minute, so that the starting template can be fully unchained, and if the high temperature treatment time is too long, it will affect the activity of the enzyme; for simple templates, pre-denaturation can also be used for 20 s, and the optimal pre-denaturation time can be determined according to the template situation. (2) It is recommended to use two-step PCR program, the annealing temperature should be 58-64℃ as the reference range, and the annealing temperature can be increased in case of non-specific reaction. If you can't get good results due to the use of primers with low Tm value, you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference. The annealing and extension times for several common instruments are shown in the following table: 20 s for Roche, BioRad, Agilent, Hongshi, Dongshenglong, etc. 30 s for ABI 7000/7300/7500. The annealing/extension times can be set according to the different types of instruments and templates, please follow the instructions of the instruments. The annealing/extension time can be set according to different models of instruments and templates, please follow the instruction manual of the instrument.
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