E. coli DNA Ligase

• Ligates DNA at nicks and cohesive termini
• Requires NAD⁺

E. coli DNA ligase catalyzes the phosphodiester bond formation between an adjacent 5ʹ phosphate and a 3ʹ hydroxyl of DNA ends, requiring NAD⁺ and Mg²⁺ as cofactors. Ligation of blunt ended DNA is extremely inefficient relative to cohesive DNA end ligation and nick sealing.

This enzme 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.

10X E. coli DNA Ligase Reaction Buffer (B6090) contains the following: 300 mM Tris-HCl, 40 mM MgCl₂, 260 µM NAD, 10 mM DTT and 0.5 mg/ml BSA; pH 8.0 at 25°C.

SDS available on request..

Enzyme properties

• Storage temperature: –25°C to –15°C

OEM by QIAGEN offers bulk manufacturing of E. coli DNA Ligase in custom formulations.

Reference

Lehman I.R. (1974) Science 186, 790-797.

Source of recombinant enzyme protein
The protein is produced by a plasmid containing the gene encoding E. coli DNA Ligase in E. coli.

Unit definition: 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.

Molecular weight: 73,606 Daltons

Quality control analysis

Protein concentration is determined by OD₂₈₀ 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 mass of the band 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.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• cDNA cloning by replacement synthesis