本公司生产的末端脱氧核糖核酸转移酶 (5U/μl)即 Terminal Deoxynucleotidyl Transferase，简称 TdT。是一种非模板依赖的 DNA 聚合酶，可以 催化在寡核苷酸、单链或双链 DNA 的 3'羟基端加上 dNTP。可以催化的寡核苷酸的最短长度为 3 个核苷酸。有报道 TdT 也可以在 RNA 的 3'羟基端 加上 NTP，但对于 RNA 的催化活性要弱于 DNA。
组分和说明
| T751007 | Component | 500U | 5×500U | Storage |
| T751007A | TdT (5U/μl) | 100μl | 5×100μl | -20℃. Avoid freeze/thaw cycle. |
| T751007B | Reaction Buffer (10×) | 0.3ml | 5×0.3ml | -20℃. Avoid freeze/thaw cycle. |
| T751007C | CoCl₂
| 0.3ml | 5×0.3ml | -20℃. Avoid freeze/thaw cycle. |
产品应用
寡核苷酸或 DNA 3'羟基末端标记；DNA 末端加尾(DNA tailing)；5'-RACE；合成同一种脱氧核苷酸的寡聚链等。
产品优势
可以催化 DNA 的 3´ 末端添加同聚物，能利用修饰碱基（如 ddNTP、DIG-dUTP）标记 DNA 3´ 末端，用于TUNEL检测（细胞凋亡的原位检测），和 TdT依赖的PCR。
使用说明
1. DNA 3'末端标记：
a. 参考如下表格设置反应体系：
| 待标记 DNA | 10 pmol of 3'-termini |
| Reaction Buffer(10×) | 5μl |
| [α-32P]-ddATP, ～10TBq/mmol(3000Ci/mmol) | 1.85 MBq(50μCi) |
| (2.5mM) CoCl₂ | 5μl |
| TdT(5U/μl) | 4μl |
| 补充无核酸酶的去离子水 | 至 50μl |
b. 按上表设置好反应体系后，轻轻混匀(可以用移液器吹打混匀或用 Vortex 在最低速度轻轻混匀)，随后离心沉淀液体。
c. 37℃孵育 20 分钟。
d. 70℃孵育 20 分钟或加入 5μl 0.5M EDTA 终止反应。
说明：标记的效率和 3'羟基末端的类型有关，3'突出末端的标记效率要显著高于 3'缩进末端或平末端的标记效率。
2. DNA 末端加尾(DNA Tailing)：
a. 参考如下表格设置反应体系：
| DNA 片断 | 1 pmol of 3'-termini |
| Reaction Buffer(10×) | 2μl |
CoCl₂
| 2μl |
| dATP or dTTP | 130pmol(20 μCi) |
| 或 dGTP or dCTP(四种中通常只需加入一种)(3000 Ci/mmol) | 60pmol |
| TdT(5U/μl) | 1.5μl |
| 补充无核酸酶的去离子水 | 至 20μl |
b. 按上表设置好反应体系后，轻轻混匀(可以用移液器吹打混匀或用 Vortex 在最低速度轻轻混匀)，随后离心沉淀液体。
c. 37℃孵育 15 分钟。
d. 70℃孵育 15 分钟或加入 5μl 0.5M EDTA 终止反应。
说明：在上述反应条件下，每个 3'羟基末端可以加上 100-130 个 dA 或 dT，或 20-30 个 dC 或 dG。
保存条件：
-20℃保存，≤0℃运输
The Terminal Deoxynucleotidyl Transferase (5U/μl) produced by our company, also known as TdT, is a non-template-dependent DNA polymerase. It can catalyze the addition of dNTP to the 3'-hydroxyl end of oligonucleotides, single-stranded or double-stranded DNA. The shortest length of the oligonucleotide that can be catalyzed is 3 nucleotides. It has been reported that TdT can also add NTP to the 3'-hydroxyl end of RNA, but its catalytic activity on RNA is weaker than that on DNA.
Components and Instructions
| T751007 | Component | 500U | 5×500U | Storage | | T751007A | TdT (5U/μl) | 100μl | 5×100μl | -20℃. Avoid freeze/thaw cycle. | | T751007B | Reaction Buffer (10X) | 0.3ml | 5×0.3ml | -20℃. Avoid freeze/thaw cycle. | | T751007C | CoCl₂
| 0.3ml | 5×0.3ml | -20℃. Avoid freeze/thaw cycle. |
|
Product Applications
Labeling the 3'-hydroxyl end of oligonucleotides or DNA; DNA tailing; 5'-RACE; synthesis of oligomeric chains of the same deoxynucleotide, etc.
Product Advantages
It can catalyze the addition of homopolymers to the 3' end of DNA, and can use modified bases (such as ddNTP, DIG-dUTP) to label the 3' end of DNA, which is applicable for TUNEL assay (in situ detection of cell apoptosis) and TdT-dependent PCR.
Instructions for Use
1. 3' end labeling of DNA:
a. Set up the reaction system with reference to the following table:
DNA to be labeled
| 10 pmol of 3'-termini | | Reaction Buffer(10X) | 5μl | | [α-32P]-ddATP, ～10TBq/mmol(3000Ci/mmol) | 1.85 MBq(50μCi) | | (2.5mM) CoCl₂ | 5μl | | TdT(5U/μl) | 4μl | Supplement with nuclease - free deionized water
| to 50μl |
|
b. After setting up the reaction system as per the above table, mix gently (by pipetting up and down or using a vortex mixer at the lowest speed), then centrifuge to precipitate the liquid.
c. Incubate at 37°C for 20 minutes.
d. Terminate the reaction by incubating at 70°C for 20 minutes or adding 5μl of 0.5M EDTA.
Note: The labeling efficiency is related to the type of 3'-hydroxyl end. The labeling efficiency of 3' overhang ends is significantly higher than that of 3' recessed ends or blunt ends.
2. DNA Tailing:
a. Set up the reaction system with reference to the following table:
| DNA fragment | 1 pmol of 3'-termini | | Reaction Buffer(10X) | 2μl | CoCl₂
| 2μl | | dATP or dTTP | 130pmol(20 μCi) | | or dGTP or dCTP(Usually only one of the four needs to be added) (3000 Ci/mmol) | 60pmol | | TdT(5U/μl) | 1.5μl | Supplement with nuclease - free deionized water
| to 20μl |
|
b. After setting up the reaction system according to the above table, mix gently (by pipetting up and down or using a vortex mixer at the lowest speed), then centrifuge to precipitate the liquid.
c. Incubate at 37°C for 15 minutes.
d. Terminate the reaction by incubating at 70°C for 15 minutes or adding 5μl of 0.5M EDTA.
Note: Under the above reaction conditions, 100-130 dA or dT, or 20-30 dC or dG can be added to each 3'-hydroxyl end.
Storage Conditions:
Store at -20°C and transport at ≤0°C.
Precautions:
(1) When using the enzyme, it should be placed on an ice box or in an ice bath. After use, it should be immediately stored at -20°C.
(2) This product is only for scientific research by professionals. It must not be used for clinical diagnosis or treatment, nor for food or drugs, and must not be stored in ordinary residences.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.
ZJ26F1039866
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