Taq DNA Polymerase 简称 Taq 酶，是最常用的 DNA 聚合酶之一。 Taq DNA Polymerase 是一种来源于嗜热菌 Thermus aquaticus 的高度热稳定的 DNA 聚合酶，分子量是 94kDa，是将 Thermus aquaticus DNA Polymerase 的基因重组表达后，分离提取而得到的。使用本制品扩增得到的 PCR 产物的 3’端附有一“A”碱 基，因此可直接克隆于 T-Vector 中。（缓冲液中镁离子浓度为 2 mM）。
组分表
FP1508912 | 组分 | 1KU | 10KU | 存储温度 | FP1508912A | Taq DNA Polymerase (5U/μL) | 200 μL | 2×1.0 mL | -20℃ | FP1508912B | 5×Taq Buffer (Mg²⁺ Plus) | 4×1.0 mL | 40 mL | -20℃ |
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适用范围
热启动法进行 PCR 扩增
注意事项
引物设计（描述引物设计时需要注意的内容）
例：
1. 引物 3'端最后一个碱基最好为 G 或者 C；
2. 引物 3'端最后 8 个碱基应避免出现连续错配；
3. 引物 3'端应避免出现发夹结构；
4. 正向引物和反向引物的 Tm 值相差不超过 1℃为佳，Tm 值调整 至 55 ~ 65℃为佳(引物 Tm 值推荐使用 Primer Premier 5 进行计算)；
5. 引物额外附加序列，即与模板非配对序列，不应参与引物 Tm 值计算；
6. 引物的 GC 含量控制在 40% ~ 60%之间；
7. 引物 A、G、C、T 整体分布要尽量均匀，避免使用 GC 或者 AT 含量高的区域；
8. 引物内部或者两条引物之间避免有 5 个碱基以上的互补序列， 两条引物的 3'端避免有 3 个碱基以上的互补序列；
9. 引物设计完毕请使用 NCBI BLAST 功能检索引物特异性，以避 免非特异性扩增产生。
使用方法
反应体系配制前准备：
a. 室温（20±5℃）/2~8℃溶解并上下颠倒混匀反应所需的各 种溶液。并放置于冰浴上或冰盒内。建议反应液体分装使用， 避免反复冻融。
b. 参考下表设置 PCR 反应体系,建议 PCR 反应体系的配制在冰 浴或在冰盒上进行：
| 组分 | 加入体积 | 终浓度 |
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| 5×Taq Buffer（Mg²⁺ Plus） | 10 μL | 1× | | dNTPs（2.5 mM each） | 0.4 μL | 0.2 mM | | Forward Primer（10 μM） | 1 μL | 0.2 μM | | Reverse Primer（10 μM） | 1 μL | 0.2 μM | | Taq DNA Polymerase（5U/μl） | 0.5 μl | 2.5U/50μl | | Template DNA* | 10 μl | / | | ddH₂O | To 50 μL | / | | 终体积 | 50 μL |
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*对于不同类型的模板在 50µl 反应体积中推荐用量如下：
哺乳动物基因组 DNA: 0. 1~ 1 µg;
大肠杆菌基因组 DNA: 10~ 100 ng;
质粒 DNA: 0. 1~ 10 ng;
c. 配制过程中应避免剧烈震荡，用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒。
反应程序
a. 常规定性 PCR（以扩增 1kb 目的片段为例）：
b. PCR 反应的设置需根据模板、引物、PCR 产物的长度和 GC 含量等条件的不同设定不同的 PCR 反应条件包括温度、时间和循环数等。
c. STEP4 (延伸) 的时间设置需根据 PCR 产物的长度进行设置，通常每 kb 产物的延伸时间为 1 min。例如 PCR 产物的长度为 1kb，则延伸时间可以设置为 1min，PCR 产物的长度为 2kb，则延伸时间可以设置为 2 min，以此类推。
d. 对于初次进行的 PCR，为尽量确保可以扩增出预期的 PCR 产物，可以把循环数设置为 35。对于需进行半定量或定量的 PCR 反应循环数一定要进行适当优化，使 PCR 反应没有达到平台期。
Taq DNA Polymerase, abbreviated as Taq enzyme, is one of the most commonly used DNA polymerases. It is a highly thermostable DNA polymerase derived from the thermophilic bacterium Thermus aquaticus, with a molecular weight of 94 kDa. The enzyme is obtained through recombinant expression of the Thermus aquaticus DNA Polymerase gene, followed by separation and purification. PCR products amplified using this product have an additional "A" base at the 3’ end, allowing direct cloning into T-Vectors (the magnesium ion concentration in the buffer is 2 mM).
Component List
FP1508912 | Components | 1KU | 10KU | Storage | FP1508912A | Taq DNA Polymerase (5U/μL) | 200 μL | 2×1.0 mL | -20℃ | FP1508912B | 5×Taq Buffer (Mg²⁺ Plus) | 4×1.0 mL | 40 mL | -20℃ |
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Application Scope
PCR amplification using the hot-start method
Precautions
Primer Design (descriptions of key considerations for primer design)Examples:
- The last base at the 3' end of the primer is preferably G or C.
- The final 8 bases at the 3' end of the primer should avoid consecutive mismatches.
- The 3' end of the primer should avoid forming hairpin structures.
- The Tm values of the forward and reverse primers should preferably differ by no more than 1℃, and the optimal Tm value range is 55~65℃ (Primer Premier 5 is recommended for calculating primer Tm values).
- Additional sequences of the primer, i.e., sequences not paired with the template, should not be included in the Tm value calculation.
- The GC content of the primer should be controlled between 40% and 60%.
- The overall distribution of A, G, C, and T in the primer should be as uniform as possible, avoiding regions with high GC or AT content.
- Avoid complementary sequences of more than 5 bases within a single primer or between the two primers; complementary sequences of more than 3 bases at the 3' ends of the two primers should also be avoided.
- After primer design, use the NCBI BLAST tool to verify primer specificity to prevent non-specific amplification.
Usage Method
Preparation before reaction system preparation:
a. Thaw all required solutions at room temperature (20±5℃) or 2~8℃, mix thoroughly by inverting up and down, and place on an ice bath or in an ice box. It is recommended to aliquot the reaction solutions for use to avoid repeated freeze-thaw cycles.
b. Prepare the PCR reaction system with reference to the table below. The preparation of the PCR reaction system is recommended to be performed on an ice bath or in an ice box.
Component
| Added Volume
| Final Concentration
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| 5×Taq Buffer (Mg²⁺ Plus) | 10 μL | 1× | | dNTPs (2.5 mM each) | 0.4 μL | 0.2 mM | | Forward Primer (10μM) | 1 μL | 0.2 μM | | Reverse Primer (10μM) | 1 μL | 0.2 μM | | Taq DNA Polymerase (5U/μl) | 0.5 μl | 2.5U/50μl | | Template DNA* | 10 μl | / | | ddH₂O | To 50 μL | / | Final Volume
| 50 μL |
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*Recommended amounts of different types of templates in a 50µl reaction volume are as follows:
Mammalian genomic DNA: 0.1~1 µg;
E. coli genomic DNA: 10~100 ng;
Plasmid DNA: 0.1~10 ng;
c. Avoid vigorous shaking during preparation. Mix gently by pipetting up and down or slight vortexing, then centrifuge at room temperature for a few seconds.
Reaction Procedure
a. Conventional qualitative PCR (taking the amplification of a 1kb target fragment as an example):
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b. PCR reaction settings (including temperature, time, and number of cycles) should be adjusted according to different conditions such as template type, primers, length of PCR product, and GC content.
c. The time for STEP4 (Extension) should be set based on the length of the PCR product. Typically, the extension time is 1 min per kb of the product. For example, if the PCR product length is 1 kb, the extension time can be set to 1 min; if the product length is 2 kb, the extension time can be set to 2 min, and so on.
d. For the first-time PCR, set the number of cycles to 35 to maximize the chance of amplifying the expected PCR product. For semi-quantitative or quantitative PCR, the number of cycles must be properly optimized to ensure the reaction does not reach the plateau phase.
ZJ26F0434220
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