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.
