The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Unit Defenition: One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5´ DNA termini concentration of 0.12 µM, 300µg/ml) in a total reaction volume of 20 µl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer: (50mM Tris-HCl (pH 7.8 at 25°C), 10mM Magnesium chloride, 10mM DTT, 1mM ATP, 25 µg/ml BSA and DNA (0.1 to 1 µM in 5´ termini)).
備考T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes.
Optimal ligation occurs at 16°C.
To dilute T4 DNA Ligase that will subsequently be stored at –20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.
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 50µM.
This protein can be used in Cloning of restriction fragments and joining linkers and adapters to blunt-ended DNA.
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Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.