Taq DNA Polymerase是通过大肠杆菌表达纯化的重组酶。其基因来源于Thermusaquaticus polymerase。该蛋白分子量为94 kDa，具有5′→3′ DNA聚合酶活性和5′→3′外切酶活性，无3′→5′外切酶活性，酶延伸速度2 kb/min，可以扩增长度达5 kb的片段。扩增得到的PCR产物3′端附有一个“A”碱基，因此可直接用于T/A克隆。本产品具有延伸速度快、扩增效率高的特点，主要适用于PCR法扩增DNA片段、DNA序列测定等实验。
| T665586 | Component | 500 U | 2500 U | 10 KU | Storage | | T665586A | Taq DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | 2×1 mL | -20℃. Avoid freeze/thaw cycle. | | T665586B | 10×PCR Buffer | 1.8 mL | 5×1.8 mL | 8×5 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 10×PCR Buffer contains 15mM Mg2+. |
活性定义：
用活性化的大马哈鱼精子DNA作为模板/引物，在74℃，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；室温存放一 个月，无明显活性改变。
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
| 试剂 | 50 μl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 | | Taq DNA Polymerase，5 U/μl | 0.25-0.5 μl | 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 | 94℃ | 2 min | / | | 变性 | 94℃ | 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 | | 终于延伸 | 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Taq 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。
Taq DNA Polymerase is a recombinant enzyme expressed and purified by Escherichia coli. Its gene is derived from the Thermus aquaticus polymer. The protein has a molecular weight of 94 kDa and exhibits 5 '→ 3' DNA polymerase activity and 5 '→ 3' exonuclease activity, without 3 '→ 5' exonuclease activity. The enzyme has an elongation rate of 2 kb/min and can amplify fragments up to 5 kb in length. The amplified PCR product has an "A" base attached to its 3 'end, making it suitable for direct use in T/A cloning. This product has the characteristics of fast extension speed and high amplification efficiency, and is mainly suitable for PCR amplification of DNA fragments, DNA sequence determination and other experiments.
| T665586 | Component | 500 U | 2500 U | 10 KU | Storage | | T665586A | Taq DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | 2×1 mL | -20℃. Avoid freeze/thaw cycle. | | T665586B | 10×PCR Buffer | 1.8 mL | 5×1.8 mL | 8×5 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 10×PCR Buffer contains 15mM Mg2+. |
Activity definition:
Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.
Quality control:
After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes 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
| Reagent | 50 μlReaction system | Final concentration | | 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 | | Taq DNA Polymerase，5 U/μl | 0.25-0.5 μl | 1.25-2.5 U/50 μl | | ddH2O | up to 50 μl | / |
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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
| Step | Temperature | Time | / | | Pre denaturation | 94℃ | 2 min | / | | Denaturation | 94℃ | 30 s | 25-35 cycles | | Anneal | 55-65℃ | 30 s | 25-35 cycles | | Extend | 72℃ | 30 s | 25-35 cycles | | Finally extended | 72℃ | 2 min | / |
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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. The amplification efficiency of Taq DNA Polymerase in this product 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.
ZJ25F1230110
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