T4 Polynucleotide Kinase
Cat. No.: SB-G3458-500U
Size: 500U
Description
T4 Polynucleotide Kinase, namely T4 polynucleotide kinase, derived from T4 bacteriophage and recombinantly expressed in Escherichia coli, is a polynucleotide 5'-hydroxyl kinase that can catalyze the transfer of the γ-phosphate group of ATP to the oligonucleus nucleotide chain (double-stranded or single-stranded DNA or RNA) on the 5'-hydroxyl terminus and 3'-monophosphate nucleoside, and the reaction is reversible. Also has 3'-terminal phosphatase activity, hydrolyzing 3'-phosphate groups from the 3'-phosphate termini of oligonucleotides, deoxy 3'-monophosphate nucleosides and deoxy 3'-diphosphate nucleosides. It is mainly used for DNA and RNA 5' end labeling and 5' end phosphorylation of synthetic DNA linkers.
- Source: Derived from T4 bacteriophage, recombinantly expressed by Escherichia coli.
- Definition of enzyme activity: at 37°C, the amount of enzyme required to incorporate 1 nmol [γ-32P]ATP into the acid-insoluble precipitate within 30 minutes is defined as one enzyme activity unit.
- Purity and concentration: SDS-PAGE detection purity ≥ 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 10 U/μL.
- Inactivation: The enzyme can be inactivated by incubating at 65°C for 20 minutes.
- Enzyme storage buffer: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4.
- 10х Reaction buffer: 500 mM Tris-HCl, 100 mM MgCl2 , 5 mM DTT, pH 7.6.
Storage and Handling Conditions
Transport with wet ice; Store at -20℃, valid for 12 months.
Composition
| Component Number |
Component |
G3458-500U |
| G3458-1 |
T4 Polynucleotide Kinase |
50 μL |
| G3458-2 |
10 × ALP Reaction Buffer |
500 μL |
| Product Manual |
1 copy |
Steps
Phosphorylation of DNA or RNA 5' ends:
1. Configure the reaction system according to the following table
| Component |
Volume |
| DNA or RNA to be phosphorylated |
200 pmol |
| 10х Reaction buffer |
5 μL |
| ATP (10 mM) |
5 μL |
| T4 Polynucleotide Kinase |
1 μL |
| Nuclease-Free Water |
To 50 μL |
2. Mix the reaction system well and incubate at 37°C for 30 min.
3. After the reaction, the system was incubated at 65°C for 20 minutes to terminate the reaction.
Precautions
1. T4 DNA ligase buffer contains 1mM ATP, so T4 Polynucleotide Kinase can co-buffer with T4 DNA ligase.
2. All enzymes should be stored in an ice box and stored at -20°C immediately after use.
3. For your safety and health, please wear a lab coat and disposable gloves for operation.
