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:Recombinant protein corresponding to T4 DNA Ligase, expressed in E. coli lambda lysogen NM 989.
Unit Definition:1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lamda DNA (5’ DNA termini concentration of 0.12uM, 300ug/ml) in a total reaction volume of 20ul in 30 minutes at 16°C 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°C.
Activity:One Weiss unit is equivalent to ~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 50X 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), hexamine chloride or by reducing the ATP concentration to 50uM.
Heat Inactivation:T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes.
T4 DNA Ligase Reaction Buffer:50mM Tris-HCl, pH 7.8 at 25°C, 10mM magnesium chloride, 10mM DTT, 1mM ATP, 25ug/ml BSA.
Reaction Conditions:1X T4 DNA Ligase Reaction Buffer and DNA (0.1-1uM in 5´ termini). Incubate at 16°C.
Applications:Suitable for use in Cloning of restriction fragments, and joining linkers and adapters to blunt-ended DNA. Other applications not tested.
Recommended Dilution:Optimal dilutions to be determined by the researcher.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
仕様
Size：20KU
Source Antigen：Recombinant, E. coli lambda lysogen NM 989
Grade：Purified
Purity：Purified
Form：Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 50mM potassium chloride, 0.1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
