ELISA GB-Clone™ Fast T4 DNA Ligase

GB-Clone™ FAST T4 DNA Ligase is designed for the efficient ligation of cohesive-ended DNA inserts into plasmid vectors in just 5-10 minutes (blunt-ended inserts in as little as 15-30 minutes). FAST T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended configuration. The enzyme has also been shown to catalyze the joining of RNA to either a DNA or RNA strand in a duplex molecµLe but will not join single-stranded nucleic acids. Biological Source: E. coli strain expressing a recombinant clone. MolecµLar Weight: 68kDa. Requirements: Mg2+, ATP and DTT. The optimum concentration of Mg2+ is 10mM. Mn2+ may be substituted for Mg2+ but is only 25% as effective as Mg2+. Inhibition: 50% inhibition by greater than 150mM NaCl (activity measured at nicks. Other inhibitors include 0.2M K+, Cs+, Li+, NH4+ and 1mM spermine. Inactivation: Heat to 70°C for 10 minutes. Contents: FAST T4 DNA Ligase (400 U/μl) 2 X Ligation Buffer We recommend that 0.5-1 μl of the ligase is used per 10 μl ligation reaction. Applications  Cloning of restriction fragments.  Joining linkers and adapters to blunt-ended DNA Unit Definition One unit of FAST T4 DNA Ligase is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of λ DNA (5´ DNA termini concentration of 0.12 μM, 300- μg/mL) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1x T4 DNA Ligase Reaction Buffer. Storage： Store at -20℃ Storage Buffer: 50 mM KCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/mL BSA and 50% glycerol. 2X T4 DNA Ligase Buffer: 60mM Tris-HCl (pH 7.8), 20mM MgCl2, 20mM DTT, 2mM ATP and 10% PEG. Physical Purity: The purity is ≥90% as judged by SDS-polyacrylamide gel electrophoresis with Coomassie® blue staining. Standard Applications We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment. Protocol: The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA. 1. Assemble the following reaction in a sterile microcentrifµge tube: vector DNA 100ng insert DNA 17ng 2 X Ligation Buffer 5μl T4 DNA Ligase 0.5–1μl Nuclease-Free Water to final volume of 10μl 2. Incubate the reaction at room temperature for 5-10 minutes for cohesive-ended ligations, or 15-30 minutes for blunt-ended ligations. Notes: 1. Ligation reactions performed using the 2 X Ligation Buffer do not need to be cleaned up before transformation. 2. Concatamers may form as ligation products. The extent of concatamer formation depends on the vector:insert ratio, incubation temperature and incubation time. This shoµLd be taken into account when screening transformants.