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.
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