AmpTherm DNA Ligase is a thermostable DNA ligase that catalyzes the formation of a phosphodiester bond between a 5´-phosphate and a 3´-hydroxyl group of two adjacent oligonucleotides hybridized to the same complementary target DNA strand. This ligation reaction occurs only when the two oligonucleotides are perfectly matched to the complementary target DNA and are directly adjacent to each other without any gaps. Therefore, it can be used for the detection of single-base substitutions. This enzyme requires ATP as a cofactor and exhibits activity over a temperature range of 45–70°C. Product information Catalog Number 14959ES84 / 14959ES92 Size 2,000 U / 10,000 U Gene Source Thermococcus sp. Expression Host E.coli Activity Definition One unit of activity (U) is defined as the amount of enzyme required to ligate 50% of 1 μg of BstEII-digested λDNA fragments (with 12 bp cohesive ends) in a 15-minute incubation at 45°C. Storage Buffer 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 200 µg/mL BSA, 50% Glycerol, 10 mM (NH4)2SO4, pH 7.4 @ 25°C Concentration 40 U/µL Component information Component No. Component Name 14959ES84 14959ES92 14959-A AmpTherm DNA Ligase (40 U/µL) 50 µL 250 µL 14959-B 10× AmpTherm DNA Ligase Buffer 200 µL 1 mL Applications 1. Allele-specific detection via Ligase Detection Reaction (LDR) and Ligase Chain Reaction (LCR). 2. Ligation of phosphorylated oligonucleotides in Ligase Chain Reaction (LCR). 3. Sealing of nicks in DNA strands at elevated temperatures. Storage conditions This product should be stored at -25~-15℃ for 2 years. Instructions for use 1. Reaction Setup: Component Volume 10× AmpTherm DNA Ligase Buffer 2 µL Substrate: DNA with complementary cohesive ends >8 nt 100 ng – 1 µg AmpTherm DNA Ligase (40 U/µL) 1-2 µL ddH2O To 20 µL 2. Reaction Conditions: Incubate at 45–50°C for 15 minutes. 3. Product Analysis: Analyze the products by agarose gel electrophoresis for longer double-stranded DNA substrates; use polyacrylamide gel electrophoresis for short DNA fragments (
