Description
Taq DNA Ligase catalyzes the formation of a phosphodiester bond in duplex DNA containing adjacent 5'-phosphoryl and 3'-hydroxyl termini, using NAD + as a cofactor (1). Supplied in: 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Tween-20, 50% glycerol (pH 7.5 at 25°C) Supplied with: 10X Taq DNA Ligase Buffer (B6060): 200 mM Tris-HCl, 250 mM KCl, 100 mM MgCl 2, 5 mM NAD +, 0.1% Triton X-100 (pH 7.6 at 25°C)
Storage temperature: –25°C to –15°C Molecular weight: 76.9 kDa Test Amount tested Specification Purity n/a >99% Specific activity n/a 400,000 U/mg Single-stranded exonuclease 400 U <5.0 % released Double-stranded exonuclease 400 U <1.0 % released Double-stranded endonuclease 400 U No conversion E. coli DNA contamination 400 U <10 copies
The enzyme is produced by a recombinant E. coli strain carrying the cloned Taq DNA Ligase gene. One unit is defined as the amount of Taq DNA Ligase required to join 50% of 1 µg of the 12-base cohesive ends of Lambda DNA cut with SmaI and SalI in 50 µL 1X Taq DNA Ligase Buffer following a 10-minute incubation at 45°C.
Usage Instructions Nick ligation in double-stranded DNA Set up the following reaction mixture in a total volume of 50 µL: Components Final Concentration Volume Nuclease-free water N/A X µL 10X Taq DNA Ligase Buffer (B6060) 1X 5 µL DNA up to 1µg X µL Taq DNA Ligase (L6060L) 80 U 2 µL Total Volume = 50 µL Incubate at 45°C for 10 minutes. Quality Control Unit activity is measured using a 2-fold serial dilution method. Dilutions of the enzyme batch were made in 1X Taq DNA Ligase Reaction Buffer and added to 50 µL reactions containing λ Hind III digested DNA and 1X Taq DNA Ligase Reaction Buffer. Reactions are incubated for 10 minutes at 45°C, stopped, and analyzed on a 0.8% agarose gel stained with ethidium bromide. Protein concentration (OD 280) 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 mass of the protein of interest band 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 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 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 16S rDNA Contamination is evaluated using 5 µL replicate samples of enzyme solution 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.
