产品内容
Component | F665641 500 U | F665641 2500 U | FastPrime DNA Polymerase, 5 U/μl | 100 μl | 5×100 μl | 10×PCR Buffer | 1.8 ml | 5×1.8 ml |
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产品简介
FastPrime DNA Polymerase 是抗Taq酶单克隆抗体和具有高效扩增及保真性能的Taq
DNA Polymerase的混合制品，适用于HOT Start PCR。使用FastPrime DNA Polymerase
进行PCR扩增时，高温变性前由于Taq酶抗体与Taq酶结合抑制DNA聚合酶活性，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。Taq酶
抗体在PCR反应最初的DNA变性步骤中变性，DNA聚合酶活性恢复，达到热启动效果。
使用本品无需特殊的对Taq酶抗体失活处理，可以在常规PCR反应条件下使用。
FastPrime DNA Polymerase具有5′→3′ DNA聚合酶活性、5′→3′ 外切酶和3′→5′
外切酶活性。与Taq DNA Polymerase相比，FastPrime DNA Polymerase具有扩增效
率高、错配率低的优良性能，能高效率扩增DNA 片段。使用本品扩增得到的PCR产物
3′ 端附有一个“A”碱基，可直接用于T/A克 隆。本产品适用于常规PCR反应和对高保真
性有要求的基因克隆等反应。
活性定义
用活性化的大马哈鱼精子DNA作为模板/引物，在74°C，30分钟内，将10 nmol脱氧
核苷酸掺入到酸性不溶物质所需的酶量定义为1个活性单位（U）。
质量控制
经过多次柱纯化，SDS-PAGE检测其纯度大于99%；经检测无外源核酸酶活性；
PCR方法检测无宿主残余DNA；能有效地扩增人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
试剂 | 50μl反应体系 | 终浓度 | 10× PCR Buffer | 5 μl | 1× | dNTP Mix，10 mM each | 1 μl | 200 μM each | Forward Primer，10 μM | 2 μl | 0.4 μM | Reverse Primer，10 μM | 2 μl | 0.4 μM | Template DNA | <0.5 μg | <0.5 μg/50 μl | FastPrime DNA Polymerase,5U/μl | 0.25-0.5 μ | 1.25-2.5 U/50 μl | ddH2O | up to 50 μl | |
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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
步骤 | 温度 | 时间 | | 预变性 | 95℃ | 2 min | | 变性 | 95℃ | 30 s | 25-35 个循环 | 退火 | 55-65℃ | 30 s | 25-35 个循环 | 延伸 | 72℃ | 30 s | 25-35 个循环 | 终延伸 | 72℃ | 2 min | |
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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，本产品的扩增效率为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。
Products content
Note: The 10 x PCR Buffer for this product contains 15 mM magnesium ions.
Products Introduction
FastPrime DNA Polymerase is a mixture of anti-Taq enzyme monoclonal antibody and Taq DNA Polymerase with high amplification and fidelity properties for HOT Start PCR. When using FastPrime DNA Polymerase for PCR amplification, Taq enzyme antibody binds to Taq enzyme and inhibits DNA polymerase activity before denaturation at low temperature. When using FastPrime DNA Polymerase for PCR amplification, before denaturation at high temperature, Taqase antibody binds to Taqase to inhibit DNA polymerase activity, which can effectively inhibit the non-specific annealing of primers and non-specific amplification caused by primer dimer at low temperature, and the Taqase antibody denatures during the initial DNA denaturation step of the PCR reaction, and the activity of DNA polymerase is restored, thus achieving the effect of Hot Start. No special inactivation of the Taqase antibody is required for the use of this product, and it can be used under conventional PCR reaction conditions.
FastPrime DNA Polymerase has 5′→3′ DNA polymerase activity, 5′→3′ exonuclease and 3′→5′ exonuclease activity. Compared with Taq DNA Polymerase, FastPrime DNA Polymerase has high amplification efficiency and low mismatch rate, and can amplify DNA fragments efficiently. The PCR products amplified with this product have an "A" base at the 3′ end, which can be directly used for T/A cloning. This product is suitable for routine PCR reactions and gene cloning reactions that require high fidelity.
Definition of activity
The amount of enzyme required to dope 10 nmol of deoxyribonucleotide into acid-insoluble material was defined as 1 activity unit (U) at 74°C for 30 min, using activated salmon sperm DNA as template/primer.
quality control
After several column purification, its purity is more than 99% by SDS-PAGE; no exogenous nuclease activity is detected; no host residual DNA is detected by PCR method; it can effectively amplify single-copy genes in the human genome.
Usage
The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment of human genomic DNA as a template, which should be improved and optimized according to the template, primer structure and size of the target fragment in actual operation.
1. 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 concentration can be increased; if a non-specific reaction occurs, the primer concentration can be decreased to optimize the reaction system.
2.PCR reaction conditions
Attention:
1）In general, the annealing temperature is 5°C lower than the melting temperature of the amplification primer, Tm. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when non-specific reaction occurs, the annealing temperature should be increased, so as 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 this product is 2 kb/min.
3) 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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