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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
Tth DNA 聚合酶是一种热稳定性酶,分子量94kDa,用大肠杆菌重组表达从来源 Thermus thermophilus HB-8 的基因。该酶在74°C时可进行DNA复制,在95°C的半衰期为20min。Tth DNA 聚合酶在镁离子存在的条件下可催化核苷酸沿5`→3`方向发生聚合反应,形成双链DNA,也可在锰离子存在的条件下,以RNA为模板沿5`→3`方向发生核苷酸聚合反应。该酶也具有5`→3`外切酶活性。Tth DNA 聚合酶可用于PCR、RT-PCR、逆转录和较高温度条件下的引物延伸反应。
组分与储存:
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适用范围:
PCR扩增、RT-qPCR扩增。
单位定义:
在 70°C 条件下,30min 内催化 10nmol dNTP 掺入到 TCA 不溶性物质所需的酶量为一个单位。
注意事项:
1. 引物设计室温/4°C溶解并混匀反应所需的各种溶液。并放置于冰浴上或冰盒内。
2. 建议反应液体分装使用,避免反复冻融。
3. 过多的模板DNA容易导致非特异性的PCR产物。
使用方法:
体系配置:
| 组分 | 加入体积 | 终浓度 |
| 2×Tth One Step RT-PCR Buffer | 25μl | 1× |
| RNasin (40U/μl) | 1μl | / |
| Forward Primer (10μM) | 1μl | 0.5μM |
| Reverse Primer (10μM) | 1μl | 0.5μM |
| Probe (10μM) | 0.25μl | 0.5μM |
| Mn²⁺ (50×) | 1μl | 1× |
| Tth DNA Polymerase | 1μl | 0.1U/μ1 |
| Template DNA | 5μl | / |
| ddH₂O | To 50μl | / |
| 终体积 | 50μl | / |
反应程序:
| 步骤名称 | 温度 | 时间 | 循环 |
| 热激 | 95℃ | 20min 30s | 1 |
| 退火 | 60℃ | 30s | 1 |
| 热激 | 95℃ | 20min 30s | 1 |
| 变性 | 94℃ | 15s | 40 |
| 退火 | 55℃ | 40s | |
| 保存 | 4℃ | ∞ | 1 |
Tth DNA polymerase is a thermostable enzyme with a molecular weight of 94 kDa. It is recombinantly expressed in Escherichia coli from the gene of Thermus thermophilus HB‑8. The enzyme performs DNA replication at 74 °C and has a half‑life of 20 min at 95 °C. In the presence of magnesium ions, Tth DNA polymerase catalyzes the polymerization of nucleotides in the 5′→3′ direction to form double‑stranded DNA. In the presence of manganese ions, it can also carry out nucleotide polymerization in the 5′→3′ direction using RNA as a template. This enzyme also possesses 5′→3′ exonuclease activity. Tth DNA polymerase can be applied to PCR, RT‑PCR, reverse transcription, and primer‑extension reactions at elevated temperatures.
Contents & Storage:
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Scope of application:
PCR amplification, RT‑qPCR amplification.
Unit definition:
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol dNTPs into TCA‑insoluble material within 30 min at 70 °C.
Precautions:
1. Dissolve and mix all reaction‑required solutions at room temperature or 4 °C, then place them on an ice bath or in an ice box.
2. It is recommended to aliquot the reaction mixture to avoid repeated freeze‑thaw cycles.
3. Excessive template DNA is liable to produce non‑specific PCR products.
Procedure:
Reaction System Setup:
| Component | Volume Added | Final Concentration |
| 2×Tth One Step RT-PCR Buffer | 25μl | 1× |
| RNasin (40U/μl) | 1μl | / |
| Forward Primer (10μM) | 1μl | 0.5μM |
| Reverse Primer (10μM) | 1μl | 0.5μM |
| Probe (10μM) | 0.25μl | 0.5μM |
| Mn²⁺ (50×) | 1μl | 1× |
| Tth DNA Polymerase | 1μl | 0.1U/μ1 |
| Template DNA | 5μl | / |
| ddH₂O | To 50μl | / |
| Final Volume | 50μl | / |
Reaction procedure:
| Step Name | Temperature | Time | Cycles |
| Heat Activation | 95℃ | 20min 30s | 1 |
| Annealing | 60℃ | 30s | 1 |
| Heat Activation | 95℃ | 20min 30s | 1 |
| Denaturation | 94℃ | 15s | 40 |
| Annealing | 55℃ | 40s | |
| Hold | 4℃ | ∞ | 1 |
| Component No. | 组分名称 | 物理外观 | 50μL | 250μL | 1ml | 储存 |
| T292976A | Tth DNA 聚合酶 | 液体 | 50μL | 250μL | 1ml | -20℃. |
| T292976B | 2×Tth 一步法反转录‑PCR 缓冲液 | 液体 | 1.25ml | 6.25ml | 25ml | -20℃. |
| T292976C | 锰离子溶液 (50×) | 液体 | 50μL | 250μL | 1ml | -20℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
| 分析证书 | T292976 |
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