Synonyms    DNA ligase 4, EC 6.5.1.1, DNA ligase IV, Polydeoxyribonucleotide synthase [ATP] 4.

Description    T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' -phosphate and 3' -hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.

Source    Escherichia Colilambda lysogen NM 989.

Physical Appearance    Sterile filtered liquid formulation having a concentration of 167,000 U/ml.

Formulation    50mM Tris-HCl (pH 7.8 at 25℃), 10mM MgCl2, 10mM DTT, 1mM ATP, 25 ?g/ml BSA and DNA (0.1 to 1 ?m in 5´ termini). ?ptimal ligation occurs at 16C.

Storage Buffer    50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 ?g/ml BSA and 50% glycerol. Store at -20C.

Stability    Two years when stored at –20℃, 2 weeks at 4℃.

Unit Defenition    1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 ul in 30 minutes at 16℃ in 1X T4 DNA Ligase Reaction Buffer. 2.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℃.

Biological Activity    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 is > 200mM.

·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 4000 (10% w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50?M.

·To dilute T4 DNA Ligase that will subsequently be stored at –200C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.

Heat Inactivation    T4 DNA Ligase can be inactivated by incubation at 65C for 10 minutes.

Quality Control    Purified free of contaminating endonucleases and exonucleases. Each lot of T4 DNA ligase is also tested in a mock cloning assay, which reveals any damage to the ligated DNA termini. Greater than 99.9% of the termini remain undamaged in this assay.

Exonuclease Activity    Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1µg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/ug) for 4 hours at 37℃ released < 0.3% of the total radioactivity.

Endonuclease Activity    Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1µg of X174 RF I DNA for 4 hours at 37℃ resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis.

Nuclease Activity    Incubation of 13,000 units for 18 hours in assay buffer (without ATP) with Hind III fragments of gamma DNA yielded a clear and sharp banding pattern on agarose gels.

Applications    Cloning of restriction fragments.