T4 DNA Ligase
T4 DNA ligase is purified from E. coli lambda lysogen NM 989.
Reagents supplied: 10x T4 Ligase buffer (w/o ATP)
Unit definition: One Cohesive-End-Unit (CEU) is defined as the amount of enzyme required to give 50% ligation of 6μg lambda DNA-Hind III fragments in a total volume of 20μl in 30 min at 16°C.
(SKUs: PD006S, PD006L)
Categories of Products
Other Products in Category
Product Details
T4 DNA ligase is purified from E. coli lambda lysogen NM 989.
Reagents supplied: 10x T4 Ligase buffer (w/o ATP)
Unit definition: One Cohesive-End-Unit (CEU) is defined as the amount of enzyme required to give 50% ligation of 6μg lambda DNA-Hind III fragments in a total volume of 20μl in 30 min at 16°C.
T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA.
Ligation Guidelines:
- Sticky-end ligation reaction:
Components | 20μl assay | Final Concentration |
10x T4 Ligase buffer | 2μl | 1x |
10mM ATP | 2μl | 1mM |
Linear DNA vector | Variable | 50-100ng |
DNA insert | variable | 1:1-1:5 vector: insert |
T4 DNA Ligase (400u/μl) | 1 μl | 400u |
Sterile ultrapure water | Up to 20 μl | |
Incubate overnight at 16oC or for 30 min at 25oC |
- Blunt-end ligation reaction:
Components | 20μl assay | Final Concentration |
10x T4 Ligase buffer | 2μl | 1x |
10mM ATP | 2μl | 1mM |
50% w/v PEG 4000 | 2μl | 5% |
Linear DNA vector | Variable | 50-100ng |
DNA insert | Variable | 1:1-1:5 vector: insert molar ratio |
T4 DNA Ligase (400u/μl) | 1 μl | 400u |
Sterile ultrapure water | Up to 20 μl | |
Incubate overnight at 16oC or for 2h at 25oC |
10x T4 Ligase Buffer: 500 mM Tris-HCl (pH 7.8), 100 mM MgCl2, 100 mM dithiothreitol
Reaction conditions:
- 1x T4 Ligase Buffer
- 400 units T4 DNA Ligase
- 1 mM ATP
- DNA (recommended DNA concentration 0.1 to 1 μM of 5´ termini)
- Optimal ligation at 16oC
Heat inactivation: 65oC for 10 minutes
Storage conditions:
- 50 mM KCl
- 10 mM Tris-HCl (pH 7.4)
- 0.1 mM EDTA
- 1 mM dithiothreitol
- 200 μg/ml BSA
- 50% glycerol
- Store at -20oC
Quality control: Tested for the absence of endo- and exodeoxyribonucleases, ribonucleases and for the capacity to join cohesive- and blunt-ended DNA fragments. More details concerning quality controls could be found in Certificate of analysis.
Notes:
- One Cohesive-End-Ligation unit is equivalent with 0.015 Weiss units.
- T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM.
- Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt-end ligation may be enhanced by addition of PEG or hexamine chloride, or by reducing the ATP concentration to 50 μM.