T4 DNA Ligase
(Concentration 2.5 Wu/μl and 6 Wu/μl*)

*Add an H to cat.# to order the high concentration

Description: T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA.

Unit definition: One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of ³²P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37°C

Reaction conditions: 50 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP (not included) and DNA (recommended DNA concentration 0.1 to 1 μM of 5´ termini). Οptimal ligation occurs at 16^oC.

Quality control: Tested for the absence of endo- and exodeoxyribonucleases, ribonucleases and for the capacity to join cohesive- and blunt-ended DNA fragments.

Storage buffer: 50 mM KCl, 10 mM Tris-HCl  (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Heat inactivation: T4 DNA Ligase can be inactivated by incubation at 65^oC for 10 minutes.

Notes

• One Weiss unit is equivalent to circa 67 cohesive-end ligation 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.

Recommended ligation mixtures:

Reagents supplied: 10x Ligase Reaction buffer (w/o ATP)