Bacteriophage T4 derived DNA ligase catalyzes the formation of phosphodiester bonds between 3’-OH termini and 5’-P termini in duplex DNA or RNA (1). 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. T4 DNA ligase was expressed in E.coli in large quantities and highly purified. MW is 55.3 kDa.
1) Insertion of DNA fragment into a vector
2) Linker (or Adaptor) ligation with DNA fragment
Storage conditions: Store at -20°C
Formulation: 10mM Tris-HCl (pH 7.6), 50mM KCl, 0.1mM EDTA, 1mM dithiothreitol, 50% glycerol
400 U/ul, where one unit is the amount of enzyme that ligates more than 90% of 6 ug of λDNA-HindIII fragments in a 20μl mixture in 30 minutes at 16°C.
Greater than 95% protein determined by SDS-PAGE (CBB staining)
The absence of endonucleases and exonucleases was confirmed.
Reagents Supplied with Enzyme:
10 x T4 Ligase Reaction Buffer (T4-Lig): 500mM Tris-HCl (pH 7.6), 100mM MgCl2, 10 mM ATP, 100mM dithiothreitol
Data Link: UniProtKB/Swiss-Prot P00970 (DNLI_BPT4)
- Weiss B et al (1968) “Enzymatic breakage and joining of deoxyribonucleic acid.” J. Biol. Chem. 243: 4543-4555 PMID: 4879167
Flg.1 SDS-PAGE of T4 DNA ligase protein.
Flg.2 DNA ligation activity.
Ligation of Hind III fragments of λ DNA using 1 unit of T4 DNA ligase
Incubation at 16℃ for 0, 10, 20, 30, and 60 min.