E. coli DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of β-NAD between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate cohesive termini. Single-stranded nucleic acids are not substrates for this enzyme.
Applications: Second-strand cDNA synthesis (1). T4 DNA Ligase alternative when blunt-end ligation is not required (2).
Source: Purified from
E. coli 594
(Su- ) bearing λ lysogen gt4lop-11
lig+ S7 (3).
Performance and Quality Testing: Endodeoxyribonuclease and 3´ and 5´ exodeoxyribonuclease assays; ligation efficiency tested.
Unit Definition: One unit is the amount of enzyme required to give 50% ligation of
Hind III-digested λ DNA in 30 min. at 16°C in a final volume of 20 µl containing a 5´ termini concentration of 0.12 ´M (300 µg/ml).
Unit Reaction Conditions: 18.8 mM Tris-HCl (pH 8.3), 90.6 mM KCl, 4.6 mM MgCl
2 , 3.8 mM DTT, 0.15 mM λ-NAD, 10 mM (NH
4 )
2 SO
4 in 20 µ
内容和储存
E. coli DNA Ligase is supplied with a vial of 10X reaction buffer [188 mM Tris-HCl (pH 8.3), 906 mM KCl, 46 mM MgCl2 , 37.5 mM
DTT, 1.5 mM λ-NAD, 100 mM (NH4 )2 SO4 ]. Store at -20°C.
