| Description | Aladdin's T7 DNA Ligase is a T7-derived, ATP-dependent dsDNA ligase recombinantly expressed in E. coli and purified using the PerfectProtein™ Technology Platform developed by aladdin. It ligates sticky ends more efficiently than blunt ends.Unlike T3 and T4 DNA Ligases, T7 DNA Ligase catalyzes Aladdin's T7 DNA Ligase is a T7-derived, ATP-dependent dsDNA ligase recombinantly expressed in E. coli and purified using the PerfectProtein™ Technology Platform developed by aladdin. It ligates sticky ends more efficiently than blunt ends.Unlike T3 and T4 DNA Ligases, T7 DNA Ligase catalyzes the formation of a phosphodiester bond between 5' phosphate and 3' hydroxyl groups of dsDNA only, but does not efficiently ligates blunt-end dsDNA. Addition of PEG6000 over 20% to the reaction can improve the ligation of blunt-end dsDNA. However, under regular ligation conditions blunt-end DNA ligation does not occur. Therefore, T7 DNA Ligase is ideal for molecular biology applications in which both blunt and sticky ends of DNA are are present but only sticky ends needs to be joined [1,2].Please refer to Figure 1 for the performance of this product in ligating sticky ends of dsDNA.Figure 1. Ligation of sticky-end DNA fragments with T7 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 sticky-end DNA. This figure is for reference only, which may vary due to different experimental conditions.sApplication：Ligation of DNA fragments digested by restriction endonuclease, ligation of dsDNA and adapters, circularization of linear dsDNA, nick repair of dsDNA, site-specific mutagenesis, Golden Gate Assembly of DNA fragments, sticky-end specific ligation, etc.Source：Purified from E. coli with recombinant expression of DNA ligase from T7 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 30 minutes at 25°C in 1X T7 DNA Ligase Reaction Buffer.Purity: No DNA ligases other than T7 DNA Ligase, no DNA endonucleases and exonucleases, no RNase, no phosphodiesterase.Inactivation or inhibition：Ligation reactions without PEG6000 can be heat inactivated by incubation at 65℃ for 10 minutes.Precautions：ATP is a cofactor of T7 DNA Ligase, unlike E. coli DNA Ligase which uses NAD as a cofactor.T7 DNA Ligase does not effectively catalyse the ligation of blunt-end DNA fragments. For the ligation of blunt-end DNA, we recommend using the T4 DNA Ligase (, D7006).T7 DNA Ligase does not catalyze the ligation of ssDNA or RNA.The T7 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, but the activity of the T7 DNA Ligase will decrease by approximately 10-fold.We recommend using Pure™ 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 T7 DNA Ligase (3KU/µl)1µl Total Volume20µlNote 1: The molar ratio of insert DNA to linearrized vector should be 3:1.Note 2: T7 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. What is the length of sticky ends that can be ligated by T7 DNA Ligase?T7 DNA Ligase can effectively catalyse the ligation of 2bp or longer sticky ends and cannot ligate 1bp sticky ends. The T7 DNA Ligase is unable to ligate blunt-end dsDNA under typical reaction conditions. However, when the reaction contains high concentration of PEG6000 (20-30% w/v), T7 DNA Ligase also has measurable ligation activity for blunt-end dsDNA.2. Can the T7 DNA Ligase be used with PEG6000-free buffers?Yes. If PEG6000 cannot be included in the reaction, a 2X Reaction Buffer without PEG6000 can be prepared.3. What are the potential factors that can cause transformation failure when using T7 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.4. 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.5. 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.6. How much DNA should be added when using T7 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 reduce 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.References: 1. Aidan J. Doherty. J Biol Chem. 1996 May 10;271(19):11083-9.2. A J Doherty. Nucleic Acids Res. 1996 Jun 15;24(12):2281-7... Read More | Inquire | Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemReagents50 µl Reaction systemFinal concentration2×Fast Probe Mixture25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes in order to make the starting template fully unchained.(2) It is recommended to use two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out a three-step PCR amplification, annealing temperature, please use the range of 56 ℃ - 64 ℃ for as a reference for the setting... Read More | Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity.Appearance:SolidIC50& Target:Ki: 4.5 µM (Fumarate hydratase)Biological Activity:Fumarate hydratase-IN-2 sodium salt (Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity.Appearance:SolidIC50& Target:Ki: 4.5 µM (Fumarate hydratase)Biological Activity:Fumarate hydratase-IN-2 sodium salt (compound 3) is a cell-permeable and competitive fumarate hydratase inhibitor ( K i =4.5 µM) with nutrient-dependent cytotoxicity... Read More | Biochemical Test:SDS-PAGE (purity > 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 4.19 |