E. coli DNA Ligase (C007B-555734)

One unit is defined as the amount of enzyme required to ligate 50% of HindIII fragments of λ DNA (5' DNA termini concentration of 0.12μM, 300μg/ml) in a total reaction volume of 20μl in 30 minutes at 16℃.Application：Ligation of dsDNA with sticky ends; nick repair of dsDNA; cloning after the synthesis of second strand cDNA [2] .Source：Recombinant E. coli DNA ligase expressed in E. coli.Enzyme storage buffer：10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 200µg/ml BSA, 50% Glycerol (pH7.4, 25℃).Inactivation or inhibition：E. coli DNA Ligase can be inactivated by incubation at 65℃ for 20 minutes.Precautions：The ligation efficiency of this product for blunt-end fragments is extremely low. Use T4 DNA Ligase for blunt-end ligation.This product cannot be used for the ligation of ssDNA or RNA.This kit is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Set up the reaction on ice as follows:ReagentVolumeFinal ConcentrationdsDNAxμlup to 0.25μg/μl10X Reaction Buffer2μl1XE. coli DNA Ligase (10U/μl)1μl0.5U/μlNuclease-free Water(17-x)μl-Total Volume20μl-Note 1: When multiple reactions are required, prepare a master mix including all reagents except the substrate DNA and then dispense to different nuclease-free PCR tubes. Finally, add substrate DNA to each tube.Note 2: E. coli DNA Ligase should be kept on ice during the experiment.2. Mix well by pipetting or vortex gently. Centrifuge briefly to collect liquid at the bottom of the PCR tube.3. Incubate at 16℃ for 30-60 minutes.4. After incubation, immediately heat inactivate the reaction at 65℃ for 20 minutes.5. Examine the reaction products by agarose gel or polypropylene gel electrophoresis. If DNA need to be recovered from agarose gel, we recommend using the DNA Gel Extraction Kit . To purify DNA from the enzyme digestion reactions, we recommend using the PCR Clean Up Kit/DNA Purification Kit .6. For other applications, please refer to the relevant literature for E. coli DNA Ligase.FAQ:1. What is the difference between E. coli DNA Ligase and T4 DNA Ligase?Under the recommended conditions in the manual, E. coli DNA Ligase cannot ligate blunt-end DNA or RNA molecules [3].2. Can E. coli DNA Ligase be heat-inactivated?E. coli DNA Ligase can be inactivated by incubation at 65℃ for 20 minutes.3. How to choose the appropriate ligase or ligation kit?For rapid ligation of blunt- or sticky-end DNA fragments, the Rapid DNA Ligation Kit is recommended. T4 DNA Ligase is suitable for most DNA recombination reactions and can be used for sticky-end (10-minute ligation at room temperature) or blunt-end (2-hour or overnight ligation at room temperature) ligations. However, E. coli DNA Ligase is more selective for substrates and is recommended for sticky-end ligations. For ligation of ssDNA or RNA molecules, we recommend using the T4 RNA Ligase .References:1. Lehman IR. Science. 1974. 186(4166):790-7.2. Okayama H and Berg P. Mol Cell Biol. 1982. 2(2):161-70.3. Higgins NP and Cozzarelli NR. Methods Enzymol. 1979. 68:50-71.

Specification: Free from endonuclease, exonuclease, RNase, phosphatase, and other types of DNA ligase.