Hotstart HiTaq Ⅱ DNA Polymerase is a chemically modified hot-start enzyme supplied in an inactive state. This ensures the enzyme remains inactive at room temperature or low temperatures, effectively preventing non-specific amplification caused by non-specific binding between primers and templates or primer dimers at ambient temperatures. Compared with ordinary Taq DNA Polymerase, it enhances the specificity, sensitivity, and yield of DNA amplification.
After a 10-minute heat shock at 95°C prior to the reaction, the enzyme activity is partially or fully activated, and the PCR hot-start time can be adjusted according to actual usage needs. Its unique buffer system expands the enzyme's applicability, enabling amplification of fragments up to 5kb. It is mainly suitable for conventional PCR, RT-PCR, and multiplex PCR, and is particularly ideal for PCR reactions requiring high specificity.
Component List
FP1508913 | Component | 250U | 1KU | 5KU | Storage |
FP1508913A | Hotstart HiTaq Ⅱ DNA Polymerase (5U/μL) | 50μl | 200μl | 1ml | -20℃. Avoid freeze/thaw cycle. |
FP1508913B | 10×Hotstart HiTaq Ⅱ Buffer (Without Mg²⁺) | 500μl | 2ml | 10ml | -20℃. Avoid freeze/thaw cycle |
FP1508913C | 100mM MgCl₂ | 100μl | 400μl | 2ml | -20℃. Avoid freeze/thaw cycle |
Application Scope
PCR amplification using the hotstart method.
Usage Method
1. Preparation before PCR Reaction System Configuration:
1.1 Thaw and mix all required reaction solutions at room temperature or 4℃, then place them on an ice bath or in an ice box. It is recommended to aliquot the reaction liquids for use to avoid repeated freezing and thawing.
1.2 The volume of the reaction system can be adjusted proportionally according to project needs, as long as the final concentration remains consistent.
1.3 Set up the PCR reaction system with reference to the table below. It is recommended that the configuration of the PCR reaction system be performed on an ice bath or in an ice box.
Component
| Added Volume
| Final Concentration
|
|---|
| 10×Hotstart HiTaq II Buffer(Without Mg²⁺) | 5 μL | 1× | | dNTPs (25 mM each) | 0.4 μL | 0.2 mM each | | Template DNA | 10 μl* | / | | Forward Primer (10 μM) | 1 μl | 0.2 μM | | Reverse Primer (10 μM) | 1 μl | 0.2 μM | | Hotstart HiTaq II DNA Polymerase (5 U/μl) | 0.5 μl | 2.5U/50μl | | 100mM MgCl₂ | 1 μl | 2mM | | ddH₂O | To 50 μl | / | | Total | 50 μl | / |
|
* Recommended Template Dosage in a 50μl Reaction Volume for Different Template Types:
Mammalian genomic DNA: 0.1~1 μg
E. coli genomic DNA: 10~100 ng
Plasmid DNA: 0.1~10 ng
a. If the DNA template is difficult to amplify in a 25μl reaction, a 50μl reaction volume can be used for better results.
b. For samples that are hard to amplify, the enzyme amount can be appropriately increased.
c. The optimal Mg²⁺ concentration depends on the primers and template type used. In most cases, adding Mg²⁺ to a final concentration of 2 mM in the system will yield good amplification results.
d. Appropriately reducing the primer concentration helps reduce the formation of primer dimers.
e. For the negative control, simply add an equal volume of water.
f. The template amount is preferably > 10⁴ copies but < 1 μg per reaction.
1.4 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
1. Conventional Qualitative PCR (taking the amplification of a 1 kb target fragment as an example):
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a. For fragments ≤ 2 kb, the denaturation time is 30 s; for fragments > 2 kb, the denaturation time is 15 s.
b. To ensure specificity, a primer Tm value > 55 ℃ is preferred, and relevant software can be used for analysis.
Experimental Examples
Common Problems and Solutions
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