Hotstart HiTaq Ⅱ DNA Polymerase 是一种化学修饰的热启动 酶，以非活性状态提供，使得该酶在常温或低温下无活性释放，从而有效避免在常温下由引物和模板非特异结合或者引物二聚体 而产生的非特异性扩增；与普通 Taq DNA Polymerase 相比，增 强了 DNA 扩增的特异性、灵敏度及扩增产物量。
Hotstart HiTaq Ⅱ DNA Polymerase 在反应前进行 95°C 热激 10min 后，酶活性部分或全部被激活，可根据实际使用选择不用 PCR 热启动 时间。独特的缓冲体系使酶的应用更加广泛，扩增片段长达 5kb，主要适用于常规 PCR、RT-PCR 以及多重 PCR 等，特别适用于对特异性要求较高的 PCR 反应。
组分表
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 |
适用范围
热启动法进行 PCR 扩增。
使用方法
1. PCR 反应体系配制前准备：
1.1 室温/4℃溶解并混匀反应所需的各种溶液，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。
1.2 反应体系配制可根据项目需要按比例调整体积，保持终浓度 一致即可。
1.3 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 |
|---|
| 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 | / |
|
* 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。
a. 如果 DNA 体系在 25μl 的反应中难以扩增，可以使用 50μl 进行反应，将会得到更好的结果；
b. 对于一些难以扩增的样本，可适当增加酶量进行反应；
c. 最佳 Mg²⁺浓度取决于所使用的引物和模板类型，大多数情况下，体系中添加终浓度 2 mM Mg²⁺将获得良好的扩增效果；
d. 适当降低引物浓度有利于减少引物二聚体的形成；
e. 对于阴性对照，则补加等体积水即可；
f. 模板量最好＞10⁴ copies 但＜1 μg/reaction。
1.4 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
1. 常规定性 PCR（以扩增 1 增 1 kb 目的片段为例）：
a. 对于≤2 kb 片段，变性时间为 30 s；对于＞2 kb 片段， 变性 时间 15 s；
b. 为保证特异性，引物 Tm 值＞55 ℃较佳，可借助相关软件 进行分析。
实验用例
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常见问题与解决方案
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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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