T3 DNA Ligase (C007B-566396)

Aladdin's T3 DNA Ligase is a T3 phage-derived, ATP-dependent dsDNA ligase recombinantly expressed in E. coli and purified using the PerfectProtein™ Technology Platform developed by aladdin. It efficiently catalyzes the ligation of dsDNA molecules with either sticky or blunt ends, as well as the repair of nicks in dsDNA or DNA/RNA hybrids with an intact DNA strand. Therefore, T3 DNA Ligase can also can be used to generate DNA-RNA and RNA-DNA fusion products, as well as for RNA ligation to form longer RNA fragments, with intact DNA as template.T3 DNA Ligase is able to catalyse the formation of phosphodiester bonds between adjacent 5' phosphate and 3' hydroxyl groups in dsDNA and is more efficient at linking A/T protruding ends than C/G ends.Similar to T4 DNA Ligase, the addition of PEG6000 to the T3 DNA Ligase reaction significantly increases the ligation efficiency for blunt-ended dsDNA. The tolerance of T3 DNA Ligase to NaCl is twice that of T4 DNA Ligase, and it can maintain 95% activity under conditions of 1.0M NaCl or KCl. Therefore, T3 DNA Ligase is ideal choice for ligation under high ion concentrations [1,2].Please refer to Figure 1 for the performance of this product in ligating sticky and blunt ends of DNA.Figure 1. Sticky and blunt end ligation of dsDNA with T3 DNA Ligase from aladdin and Competitor. A. Colonies of DH5ɑ transformed with the ligation products; B. Colony PCR of clones randomly selected from plates in figure A. The experimental results demonstrates that this product has comparable performance with the widely received Competitor product in ligating dsDNA with either sticky ends or blunt ends. This figur e is for reference only, which may vary due to different experimental conditions.sApplication：Ligation of DNA fragments digested by restriction endonuclease, ligation of PCR products, ligation of dsDNA and adapters, circularization of linear dsDNA, nick repair of dsDNA, site-specific mutagenesis, ligation under high salt conditions, DNA-guided DNA-RNA or RNA ligation.Source：Purified from E. coli with recombinant expression of DNA ligase from T3 phage.Definition of enzyme activity unit: One unit is defined as the amount of enzyme required for 50% ligation of 100ng HindIII fragments of λ DNA in a total reaction volume of 20μl in 1 minute at 25℃ in 1X T3 DNA Ligase Reaction Buffer.Purity: No DNA ligases other than T3 DNA Ligase, no endonucleases and exonucleases, no RNase, no phosphodiesterase.Enzyme storage buffer：10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH7.4 at 25℃).Inactivation or inhibition：Ligation reactions without PEG6000 can be heat inactivated by incubation at 65℃ for 10 minutes.Precautions：ATP is a cofactor of T3 DNA Ligase, unlike E. coli DNA Ligase which uses NAD as a cofactor. The 2X Reaction Buffer supplied with this product contains the ATP and the PEG6000 required for sticky end ligation.The catalytic substrate of T3 DNA Ligase is double-stranded DNA (dsDNA) or nicked DNA/RNA with an intact DNA strand. It cannot be used for ligation of single-stranded DNA or RNA, but can be used for DNA-guided ligation of single-stranded DNA, single-stranded RNA or between single-stranded DNA and single-stranded RNA.The T3 DNA Ligase reaction contains 7.5% PEG6000. If the subsequent experiment is not compatible with PEG6000, a home-made ligation buffer without PEG6000 or the Ligation Buffer for T4 DNA Ligase (, D7006) can be used, and ATP should be added at a final concentration of 1mM. However, the activity of the T3 DNA Ligase in the Ligase Buffer for T4 DNA Ligase will decrease by approximately 10-fold.In a reaction without PEG6000, T3 DNA Ligase can be heat inactivated. But the ligation reaction containing the PEG6000 cannot be heat inactivated. Otherwise the transformation efficiency will be significantly reduced.If a high concentration of NaCl is required for the ligation, we recommend using a buffer without PEG6000.A standard 20μl reaction typically requires 30 minutes at 25℃.We recommend using PurTM Ultrapure Water (DNase/RNase-free, Sterile) (, ST876) for the reaction.This product 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 ligation reaction in a nuclease-free microfuge tube on ice as follows:ReagentVolume2X Reaction Buffer10μlVector DNAXμl (0.020pmol)Insert DNAYμl (0.060pmol)Ultrapure Water (9-X-Y)μl T3 DNA Ligase (3kU/μl)1μl Total Volume20μlNote 1: The molar ratio of insert DNA to linearrized vector should be 3:1.Note 2: T3 DNA Ligase should be added last.2. Mix well by pipetting and centrifuge briefly to collect the liquid to the bottom of the tube.3. Incubate at 25℃ (or room temperature) for 15-30 minutes.4. Chill on ice and transform 5μl of ligation product into 50μl of competent cells. The remaining sample can be stored at -20℃.Note 1: Heat deactivation cannot be performed, as it will dramatically reduce transformation efficiency.Note 2: To analyze the ligation efficiency, the reaction product can be subjected to agarose/PAGE analysis. If DNA recovery from agarose gel is required, we recommend using the DNA Gel Recovery Kit (, D0056).FAQ:1. Can the T3 DNA Ligase be used with PEG6000-free buffers?Yes. But the activity of T3 DNA ligase may be lower in such buffers.2. What are the potential factors that can cause transformation failure when using T3 DNA Ligase for ligation?a. Lack of ATP or Mg2+ in the reaction. ATP easily degrades upon repeated freeze-thaw of this product. We recommend storing this product in aliquot to avoid repeated freeze-thaw, or adding an appropriate amount of ATP in this product after being used for multiple times to ensure ligation efficiency.b. The presence of high salt or EDTA in the reaction. We recommend purifying the DNA substrate before ligation to remove the interference.c. Phosphatases such as CIP, BAP or SAP used for dephosphorylation of the DNA substrate are not completely inactivated before ligation. The phosphatases should be completely deactivated or removed according to the recommended procedures.d. Too high a concentration of DNA in the reaction can result in the production of linear DNA only. The total concentration of DNA in the ligation reaction should be within 1-10μg/ml.e. Adding too much ligation product to competent cells can cause failure of the transformation. We recommend transforming 1-5μl of ligation product to 50μl of competent cells.f. Prolonged ligation in the presence of PEG6000 can produce large DNA fragments that inhibit transformation.g. The ligation product was not purified prior to electroporation. The presence of both salt and PEG6000 has a significant impact on electroporation. We recommend purifying the ligation product using a purification column before electroporation.h. Incomplete digestion of the vector will result in lack of target clones containing the insert.3. What other factors could cause a low transformation efficiency?a. The competent cells have too low transformation efficiency. New batch of competent cells should be used.b. Whether the ligated DNA contains an E. coli antagonistic inverted repeat or a tandem repeat.c. The insert DNA of mammalian or plant origin may contain methylated cytosines that can be degraded by a wide range of E. coli strains. We recommend using mcrA, mcrBC and mrr deficient E. coli competent cells for transformation.d. The constructed plasmid that is ＞10kb should be transformed with electroporation, instead of chemical transformation. 4. What problems in restriction endonuclease digestion can lead to failure of the ligation reaction or subsequent transformation?a. The digestion is not complete. If digestion occurs at the end of a PCR fragment, there must be approximately 6 protection bases at 5' end of the recognition site. Meanwhile, test the activity of the restriction enzyme with a control substrate.b. The restriction endonuclease is not completely inactivated. If the restriction endonuclease cannot be heat inactivated, the DNA can be purified with an appropriate method.c. Restriction endonuclease generated star activity. We recommend analyzing the cleaved DNA by gel electrophoresis, reducing the amount of restriction endonuclease, or reducing the digestion time appropriately.d. DNA purification is recommended when DNA or restriction endonucleases contain nucleic acid exonucleases or phosphatases that destroy the ends of DNA fragments.5. How much DNA should be added when using T3 DNA Ligase?To improve the formation of circular DNA as well as the transformation efficiency, the total DNA concentration in the ligation reaction should be within 1-10μg/ml to ensure effective ligation. The molar ratio of insert DNA to linearised vector is recommended to be 3:1. Molar ratios lower than 2:1 will cause a reduction of ligation efficiency, while molar ratios higher than 6:1 will result in the insertion of multiple fragments. If the concentration of substrate DNA cannot be determined, multiple ratios can be tested.6. Is T3 DNA ligase more tolerant to salt ions than T4 DNA ligase?Yes. The T3 DNA Ligase is able to tolerate 250-300mM of salt. Please also note that in the presence of PEG, DNA may precipitate in high salt conditions, which can inhibit the ligation reaction.7. Can T3 DNA Ligase be heat inactivated?Yes. T3 DNA Ligase can be heat inactivated by incubation at 65℃ for 10 minutes in a reaction without PEG. However, do not heat inactivate the ligation reaction containing PEG, as it will dramatically reduce transformation efficiency. 8. What control should be used to test cells and DNA when using T3 DNA ligase?Note: Use the same DNA concentration for each control and 0.1-1.0ng of DNA for each transformation.a. Cell viability and antibiotic resistance can be assayed by transforming the undigested vector into competent cells which are then spread evenly onto plates with/without antibiotics.b. Detect whether the vector is linearised completely. The number of clones obtained from linearised vector transformation should be less than 10% of the clone number in step a.c. Use re-ligated plasmids to test ligase activity and DNA end integrity. Transform digested and modified vectors into competent cells and the number of clones obtained should be close to the number of clones in step a.d. Use the digested, phosphorylated and re-ligated plasmids to detect background caused by incomplete dephosphorylatiion. Transformation of the cleaved, phosphorylated and re-ligated plasmid into competent cells should result in less than 10% of clones from step a.References: 1. Cai L, Hu C, Shen S, Wang W, Huang W. 2004. 135(3):397-403.2. Lei Y, Washington J, Hili R. Org Biomol Chem. 2019. 17(7):1962-1965.

Specification: No DNA ligases other than T3 DNA Ligase, no endonucleases and exonucleases, no RNase, no phosphodiesterase.