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 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.