从一种携带有高表达 T4 DNA Polymerase 基因的大肠杆菌（E. coli）中提纯制备。
T4 DNA Polymerase，即 T4 DNA 聚合酶，是一种模板依赖的 DNA 聚合酶，可以在结合有引物的单链 DNA 模板上，从 5'→3' 方向催化 DNA 合成反应。T4 DNA Polymerase 具有 3'→5' 外切酶活性，但不具有 5'→3' 外切酶活性，其 3'→5' 外切核酸酶活性比 E. coli DNA 聚合酶 I 强。主要应用于 DNA 5' 或 3' 突出末端的平滑化、定点突变过程中第二链的合成以及不依赖于连接反应的 PCR 产物克隆等方向中。
组分表
FP1508928 | 组分 | 300U | 3KU | 30KU | 存储温度 |
FP1508928A | T4 DNA Polymerase (3U/μL) | 100 μL | 1.0 mL | 10 mL | -20℃ |
FP1508928B | 10 ×T4 DNA Polymerase Buffer | 1.0 mL | 10×1.0 mL | 100 mL | -20℃ |
FP1508928C | Solution I (100×) | 100 μL | 1.0 mL | 10 mL | -20℃ |
适用范围
1. 缺口填充（无链置换活性），产生平末端；
2. 补平 5' 突出端，形成平末端；
3. 切除 3' 突出末端，形成平末端；
4. 通过置换合成法标记探针；
5. 单链删除后亚克隆；
6. 基因定点突变中第二条链的合成。
注意事项
1. 由于该酶具有 3'→5' 外切酶的活性，提高反应温度、增加酶量、没有加入 dNTP 或反应时间过长都可能造成 DNA 末端碱基被切除形成凹陷。
2. 使用该酶时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于 - 20℃保存。
3. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用方法
反应体系配制前准备：
a. 4℃溶解反应所需的各种试剂，并放置于冰浴上或冰盒内使用。建议反应液体分装使用，避免反复冻融。
b. 可参考下表中的末端修复体系设置反应体系，建议在冰浴或在冰盒上进行体系配制：
组分 | 体积 | 终浓度 |
|---|
10×T4 DNA Polymerase Buffer | 5 μL | 1× | dNTP Mix* (25 mM) | 0.4 μL | 0.2 mM | Solution I (100×) | 0.5 μL | 1× | T4 DNA Polymerase (3 U/μl) | —** | — | 反应物 | —** | — | ddH₂O | To 50 μL | — | 终体积 | 50 μL | — |
|
*
10×T4 DNA Polymerase Buffer 不包含 dNTP Mix，需客户自备。
** 建议每微克 DNA 底物添加 1U 的 T4 DNA Polymerase。
反应程序（以末端修复为例）
步骤名称 | 温度 | 时间 |
|---|
反应 | 12~37℃ | 15~30 min | 灭活 | 75℃ | 20 min | 保存 | 4℃ | ∞ |
|
注：可根据具体底物用量及酶量调整反应温度及时间。
It is purified and prepared from E. coli carrying a highly expressed T4 DNA Polymerase gene.
T4 DNA Polymerase, namely T4 DNA polymerase, is a template-dependent DNA polymerase, which can catalyze DNA synthesis reaction from the 5'→3' direction on a single-stranded DNA template bound with a primer. T4 DNA Polymerase has 3'→5' exonuclease activity, but no 5'→3' exonuclease activity, and its 3'→5' exonuclease activity is stronger than that of E. coli DNA polymerase I. It is mainly used in the blunting of 5' or 3' protruding ends of DNA, the synthesis of the second strand during site-directed mutagenesis, and the cloning of PCR products independent of ligation reaction, etc.
Component List
FP1508928 | Components | 300U | 3KU | 30KU | Storage |
FP1508928A | T4 DNA Polymerase (3U/μL) | 100 μL | 1.0 mL | 10 mL | -20℃ |
FP1508928B | 10 ×T4 DNA Polymerase Buffer | 1.0 mL | 10×1.0 mL | 100 mL | -20℃ |
FP1508928C | Solution I (100×) | 100 μL | 1.0 mL | 10 mL | -20℃ |
Scope of Application
1. Gap filling (no strand displacement activity) to generate blunt ends;
2. Filling 5' overhangs to form blunt ends;
3. Trimming 3' overhangs to form blunt ends;
4. Probe labeling via displacement synthesis;
5. Subcloning after single-strand deletion;
6. Synthesis of the second strand in site-directed mutagenesis.
Precautions
1. Since this enzyme has 3'→5' exonuclease activity, increasing the reaction temperature, increasing the enzyme amount, not adding dNTPs, or prolonging the reaction time may lead to the excision of bases at the DNA ends, resulting in recessed ends.
2. When using this enzyme, it should be stored in an ice box or on an ice bath, and immediately placed at -20°C for storage after use.
3. For your safety and health, please wear a lab coat and disposable gloves during operation.
Usage Method
Preparation before reaction system preparation:
a. Dissolve various reagents required for the reaction at 4°C, and place them on an ice bath or in an ice box for use. It is recommended to aliquot the reaction solution for use to avoid repeated freeze-thaw cycles.
b. The reaction system can be set up with reference to the end repair system in the table below, and it is recommended to prepare the system on an ice bath or in an ice box:
Component
| Volume
| Final Concentration
|
|---|
10×T4 DNA Polymerase Buffer | 5 μL | 1× | dNTP Mix* (25 mM) | 0.4 μL | 0.2 mM | Solution I (100×) | 0.5 μL | 1× | T4 DNA Polymerase (3 U/μl) | —** | — | Reactants
| —** | — | ddH₂O | To 50 μL | — | Final Volume
| 50 μL | — |
|
* 10×T4 DNA Polymerase Buffer does not contain dNTP Mix and needs to be prepared by customers themselves.
** It is recommended to add 1 U of T4 DNA Polymerase per microgram of DNA substrate.
Reaction Procedure (taking end repair as an example)
Step Name
| Temperature
| Time
|
|---|
Reaction
| 12~37℃ | 15~30 min | Inactivation
| 75℃ | 20 min | Storage
| 4℃ | ∞ |
|
Note: The reaction temperature and time can be adjusted according to the specific amount of substrate and enzyme.
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