Description
E. coli DNA ligase catalyzes the formation of phosphodiester bonds between an adjacent 5ʹ phosphate and a 3ʹ hydroxyl of DNA ends, requiring NAD + and Mg 2+ as cofactors. The ligation of blunt-ended DNA is extremely inefficient relative to cohesive DNA end ligation and nick sealing. This enzyme is supplied in 10 mM Tris-HCl, 50 mM KCl. 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C. The E. coli DNA Ligase Reaction Buffer with a concentration of 10x includes 300 mM Tris-HCl, 40 mM MgCl 2, 260 µM NAD, 10 mM DTT, and 0.5 mg/mL BSA at pH 8.0 and a temperature of 25°C.
Storage temperature: –25°C to –15°C Molecular weight: 73,606 Daltons Test Units tested Specification Purity n/a >99% Specific activity n/a 20,000–28,880 U/mg Single-stranded exonuclease 100 U <2% released Double-stranded exonuclease 100 U <1% released Double-stranded endonuclease 100 U No conversion E. coli DNA contamination 50 U <10 copies Reference Lehman I.R. (1974) Science 186, 790-797.
The recombinant enzyme protein is produced by a plasmid containing the gene encoding E. coli DNA Ligase in E. coli. One unit is defined as the amount of E. coli DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 25°C.
Usage Instructions 1. Set up the following nick sealing reaction mixture in a total volume of 20 µL: 2 µL 10x E. coli DNA Ligase Buffer (B6090) Up to 5 µg of DNA 1 µL (10 U) E. coli DNA Ligase (L6090L) Nuclease-free water up to 20 µL 2. Incubate the reaction mixture at 16°C for 30 minutes. 3. Stop the reaction by heat inactivation at 65°C for 20 minutes. Quality Control Protein concentration is determined by OD 280 absorbance. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C. E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
