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FAQs for E. coli DNA Ligase FAQs for DNA Modifying Enzymes and Cloning Technical Reference for DNA Modifying Enzymes and Cloning Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE E. coli DNA Ligase Reaction Buffer

Home > Products > DNA Modifying Enzymes and Cloning > Ligases > E. coli DNA Ligase E. coli DNA Ligase Catalog # Size Concentration Price Qty   M0205L 1,000 units 10,000 units/ml $212.00 M0205S 200 units 10,000 units/ml $53.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Specific Activity: 6,000 units/mg Supplied with 10X Reaction Buffer Description: Catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA containing cohesive ends. Source: Purified from E. coli strain 594(su-) carrying the prophage λgt4 lop11 lig+Sam 7 (1) by the procedure of Panasenko et al. (2). Applications: Okayama and Berg cDNA cloning (3) Reagents Supplied: E. coli DNA Ligase Reaction Buffer (10X) Enzyme Properties Heat Inactivation: 65°C for 20 minutes Specific Activity: 6,000 units/mg Reaction & Storage Conditions Reaction Conditions: 1X E. coli DNA Ligase Reaction Buffer Incubate at 16°C. 1X E. coli DNA Ligase Reaction Buffer: 30 mM Tris-HCl 4 mM MgCl2 26 µM NAD 1 mM Dithiothreitol 50 µg/ml BSA pH 8.0 @ 25°C Unit Definition: One unit is defined as the amount of enzyme required to give 50% ligation of HindIII 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 E. coli DNA Ligase Reaction Buffer. Concentration: 10,000 units/ml Storage Conditions: 10 mM Tris-HCl 50 mM KCl 1 mM Dithiothreitol 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes General notes: Does not ligate RNA to DNA (4). This enzyme ligates only DNA fragments with cohesive termini. Usage notes: Requires NAD+ (nicotinamide adenine dinucleotide) as a cofactor, in contrast to other ligases which use rATP. Ligation of blunt-ended fragments is extremely inefficient. For ligation of blunt-ended fragments use T4 DNA Ligase. FAQs How does E. coli DNA Ligase differ from T4 DNA Ligase? Should E.coli DNA Ligase be used for cloning? When should E. coli DNA Ligase be used? Will PEG increase E. coli DNA Ligase activity? Can E. coli DNA Ligase be used at temperatures other than 16°C? Does E. coli DNA Ligase require NAD? How much DNA should be used with E. coli DNA Ligase? Is the E. coli DNA Ligase used in any special techniques? Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Exonuclease Activity: Incubation of 20 units of enzyme for 4 hours at 37°C in 50 μl assay buffer containing 1 μg sonicated E. coli 3H DNA (105 cpm/μg) gave < 0.1% acid soluble counts. 16-Hour Incubation: Incubation of 20 units of E. coli DNA Ligase with 1 μg of λ DNA (HindIII digest) for 16 hours in 50 µl of 1X NEBuffer 3 at 37°C resulted in a normal and sharp banding pattern on agarose gels. References Panasenko, S.M. et al. (1977) Science, 196, 188-189. Panasenko, S.M. et al. (1978) J. Biol. Chem., 253, 4590-4592. Okayama, H. and Berg, P. (1982) Mol. Cell. Biol., 2, 161-170. Higgins, N.P. and Cozzarelli, N.R. (1979) R. Wu (Eds.), Methods Enzymol., 68, pp. 50-71. New York: Academic Press. Reagents Sold Separately E. coli DNA Ligase Reaction Buffer