Description
T3 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5ʹ phosphate and a 3ʹ hydroxyl termini in duplex DNA. The enzyme joins blunt ends and cohesive end termini and repairs single-stranded nicks in duplex DNA. In the absence of 20–30% PEG 6000, T3 DNA Ligase displays a very low efficiency for blunt-ended ligation. T3 DNA Ligase is more efficient for joining A and T overhangs than C- and G-matched ends. T3 DNA Ligase retains 95% of its activity in 1.0 M NaCl or KCl, with an optimal concentration of 300 mM, making the enzyme recommended for reactions with increased salt concentrations. This enzyme is supplied in 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.5 at 25°C. The 2x Rapid Ligation Buffer contains the following: 132 mM Tris-HCI, 20 mM MgCl 2, 2 mM DTT, 2 mM ATP at 15% PEG 6000; pH 7.6 at 25°C.
Storage temperature: –25°C to –15°C Molecular weight: 37,350 Daltons Test Units tested Specification Purity n/a >99% Specific activity n/a 3,000,000 U/mg Single-stranded exonuclease 30,000 U <1% released Double-stranded exonuclease 30,000 U <1% released Double-stranded endonuclease 30,000 U No conversion E. coli DNA contamination 30,000 U <10 copies
The recombinant enzyme protein is produced by a recombinant E. coli strain carrying the T3 DNA Ligase gene. One unit is defined as the amount of T3 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in 30 minutes at 23°C.
Usage Instructions Sticky-end ligation of cut vector and insert. Recommended DNA molar ratio, vector: insert = 1: 3 1. Set up the following reaction mixture in a total volume of 20 µL (add the ligase last): 10 µL 2x Rapid Ligation Buffer (B1010) 50 ng vector DNA 3-fold molar excess of insert DNA 1 µL T3 DNA Ligase (L6010) Nuclease-free water up to 20 µL 2. Incubate the reaction mixture at 25°C for 15-30 minutes. 3. Leave the ligated product on the ice and transform 1-5 µL of the product into 50 µL of competent cells. Alternatively, the ligation product can be purified using the appropriate method or stored at -20°C. Notes 1. T3 DNA Ligase is supplied with 2x Rapid Ligation Buffer for use in ligation reactions. Activity is very low in the absence of PEG (1). 2. Heat inactivation reduces transformation efficiency dramatically. The enzyme has also been characterized in the 1x T3 DNA Ligase Buffer, with a 17-fold decrease in specific activity due to the absence of the crowding agent (PEG 6000). The 1x T3 DNA Ligase Buffer contains 50 mM Tris-HCl, 300 mM NaCl, 0.5 mM ATP and 1 mM DTT; pH 8.0 at 25°C. The following information was taken from the Cai reference (Cai, Liang, et al. (2004) J. Biochem. 135:397), which describes the characterization of the T3 DNA Ligase: Optimal pH: 8.0 Optimal reaction temperature: 20°C Optimal reaction time: >30 minutes (without PEG 6000) Optimal ionic strength: 300 mM NaCl Optimal divalent cation/concentration: 2 mM Mg 2+ Optimal cofactor: ATP (0.5 mM) Quality Control Unit activity was measured using a twofold serial dilution method. Dilutions of the enzyme were made in 1x Rapid Ligation Buffer and added to 20 µL reactions containing double-stranded DNA fragments and 1x Rapid Ligation Buffer. Reactions were incubated for 30 minutes at 23°C, placed on ice and analyzed on a 1% agarose gel stained with ethidium bromide. 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.
