Hotstart HiTaq DNA Polymerase is a thermostable Taq DNA polymerase modified with chemical groups. Before high-temperature heating, the chemical modification groups bind to the Taq enzyme, inhibiting its polymerase activity. This avoids non-specific amplification from primer extension or formation of primer dimers, enhancing the specificity, sensitivity, and stability of DNA amplification. It is widely applicable to conventional PCR, multiplex PCR, nested PCR, and other applications. The enzyme can recover its activity after heat shock at 95℃ for 10 minutes. In addition, no 3'→5' proofreading exonuclease activity is detected in Hotstart HiTaq DNA Polymerase, but it possesses 5'→3' exonuclease activity, making it suitable for quantitative real-time PCR (qPCR) detection. Hotstart HiTaq DNA Polymerase is inactive at room temperature, facilitating room-temperature operation of PCR experiments.
Component List
FP1509104 | Components | 250U | 1KU | 5KU | Storage |
FP1509104A | Hotstart HiTaq DNA Polymerase (5U/μL) | 50 μL | 200 μL | 1.0 mL | -20℃ |
FP1509104B | 5×HS HiTaq Buffer (Mg²⁺ Plus) | 1.0 mL | 4×1.0 mL | 20 mL | -20℃ |
FP1509104C | Solution I (10×) | 500 μL | 2×1.0 mL | 10 mL | -20℃ |
Application Scope
Suitable for hot-start PCR and qPCR.
Precautions
Primer Design
- 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
Thaw and mix all required PCR reaction solutions thoroughly, then place on an ice bath or in an ice box. It is recommended to aliquot the PCR reaction solutions for use to avoid repeated freeze-thaw cycles. Set up the PCR reaction with reference to the table below:
- Preparation of the PCR reaction system:
| Component | Added Volume | Final Concentration | | 5×HS HiTaq Buffer(Mg²⁺ Plus) | 4 μL | 1× | | Solution I（10×） | 2 μL | 1× | | dNTP（25mM each） | 0.16 μL | 0.2 mM | | Hotstart HiTaq DNA Polymerase（5U/μL） | 0.2 μL | — | | Forward Primer（10μM） | 0.4 μL | 0.2 μM | | Reverse Primer（10μM） | 0.4 μL | 0.2 μM | Template DNA*
| 4 μL(※) | — | | ddH₂O | To 20 μL | — | Final Volume
| 20 μL |
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※ Amount of Template DNA: To ensure reaction sensitivity, use 10⁴ copies of the target sequence as the template in a 20 μL reaction system. The amount of template to be added to the PCR system can be calculated with reference to the table below.
Molar Number of DNA from Various Sources per 1 μg
| 1 μg DNA | Mol |
|---|
| 1 kb linear double-stranded DNA | 9.18×10¹¹ | | 3 kb Plasmid DNA | 2.9×10¹⁰ | | Lambda (λ) DNA | 1.9×10¹⁰ | | E. coli Genomic DNA | 2.0×10⁸ | | Human Genomic DNA | 3.0×10⁵ |
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For example: Purified human genomic DNA has a concentration of 1 μg/μL. If a gene has 10 copies in the human genome, its copy number per unit volume is calculated as: 3.0×10⁵ mol/μg × 1 μg/μL × 10 copy/mol = 3.0×10⁶ copy/μL. To obtain 10⁴ copies, the volume needed is 10⁴ copy / (3.0×10⁶ copy/μL) = 1/300 μL. That is, 1/300 μL of human genomic DNA at 1 μg/μL concentration contains 10⁴ copies of the gene. Dilute it 300-fold and add 1 μL to a 25 μL PCR system. To ensure reaction specificity, the final DNA concentration should be less than 10 ng/μL; excessive DNA may cause smearing or even non-specific bands.
2. Mix gently by pipetting up and down or slight vortexing, then centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
3. Place each prepared PCR reaction tube in the PCR instrument and start the PCR reaction.
Reaction Procedure
1. Conventional Qualitative PCR (taking the amplification of a 1 kb target fragment as an example)
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2. Quantitative Real-time PCR
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a. PCR reaction conditions (including temperature, time, and number of cycles) should be set differently based on factors such as template, primers, length of PCR product, and GC content.
b. The time for STEP4 (Extension) should be set according to 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.
c. 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 reaches the plateau phase.
