T4 DNA Ligase (Fast)
Based on 1 publication(s) in Google Scholar
MCE T4 DNA Ligase (Fast) is produced by Escherichia coli carrying a T4 phage, catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA.1 U=1 Weiss unit
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Storage :
-20°C, 1 year
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Description & Advantages
MCE T4 DNA Ligase (Fast) is produced by Escherichia coli carrying a T4 phage, catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme joins DNA fragments with either cohesive or blunt termini, repairs single-strand nicks in duplex DNA, RNA or DNA/RNA hybrids, but it is inactive against single-stranded nucleic acids. It is suitable for labeling RNA 3'-ends, cyclizing RNA and DNA oligonucleotides and cloning cDNA. The T4 DNA Ligase requires ATP as a cofactor, completes viscous end connections in just 10 min at room temperature. Simple handling for fast and efficient connections.
Protocol
1. Cohesive-end ligation
| Component | 20 μL reaction |
|---|---|
| Linearized vector DNA | 20-100 ng |
| Insert DNA | Fragment : Vector molar ratio = 3 : 1-10 : 1 |
| 10× T4 DNA Ligase Buffer | 2 μL |
| T4 DNA Ligase (Fast) (5 U/μL) | 0.2 μL (1 U) |
| ddH2O | To 20 μL |
Mix gently, briefly centrifuge, and incubate at 22°C for 10 min. The ligation product may be used directly for transformation. Use 1-5 μL product for 50 μL chemically competent cells, or 1-2 μL product for 50 μL electrocompetent cells.
2. Blunt-end ligation
| Component | 20 μL reaction |
|---|---|
| Linearized vector DNA | 20-100 ng |
| Insert DNA | Fragment : Vector molar ratio = 3 : 1-10 : 1 |
| 10× T4 DNA Ligase Buffer | 2 μL |
| 50% PEG | 2 μL |
| T4 DNA Ligase (Fast) (5 U/μL) | 1 μL (5 U) |
| ddH2O | To 20 μL |
Mix gently, briefly centrifuge, and incubate at 22°C for 1 h. Proceed to transformation as described above.
3. Circularization of linear DNA
| Component | 50 μL reaction |
|---|---|
| Linear DNA | 10-50 ng |
| 10× T4 DNA Ligase Buffer | 5 μL |
| T4 DNA Ligase (Fast) (5 U/μL) | 1 μL (5 U) |
| ddH2O | To 50 μL |
Mix gently, briefly centrifuge, and incubate at 22°C for 10 min. The ligation product may be used directly for downstream transformation.
4. Linker ligation
| Component | 20 μL reaction |
|---|---|
| Linear DNA | 100-500 ng |
| Phosphorylated linker | 1-2 μg |
| 50% PEG | 2 μL |
| 10× T4 DNA Ligase Buffer | 2 μL |
| T4 DNA Ligase (Fast) (5 U/μL) | 0.4 μL (2 U) |
| ddH2O | To 20 μL |
Mix gently, briefly centrifuge, and incubate at 22°C for 10 min. If heat inactivation is required, incubate at 65°C for 10 min or at 70°C for 5 min.
Note: 1 U = 1 Weiss unit. Completely thaw and thoroughly mix 10× T4 DNA Ligase Buffer before use.
Publications
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Journal Impact Factor
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Most Recent
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Cancer Lett2025 Jul 28:623:217726. PMID: 40250791
Storage
-20°C, 1 year
Components
| Components | HY-K1049-1000U |
|---|---|
| T4 DNA Ligase (Fast) (5U/μL) | 200 μL |
| 10 × T4 DNA Ligase Buffer | 1 mL × 2 |
| 50% PEG | 1 mL |
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