T3 DNA Ligase produced by Aladdin is an ATP-dependent double-stranded DNA ligase derived from T3 bacteriophage, expressed and purified using our proprietary technical platform.T3 DNA Ligase efficiently catalyzes the ligation of cohesive-ended and blunt-ended double-stranded DNA molecules, as well as the nick repair of double-stranded DNA or DNA-RNA hybrid duplexes. In nick repair, the intact strand is DNA, and the 5'-phosphoryl and 3'-hydroxyl ends of the nicked strand can be DNA-DNA, DNA-RNA, RNA-DNA, or RNA-RNA, all of which can be repaired by T3 DNA Ligase.Therefore, T3 DNA Ligase can also be used to generate DNA-RNA and RNA-DNA fusion nucleic acids, as well as for DNA-templated RNA ligation to form long RNA fragments. T3 DNA Ligase catalyzes the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA, and exhibits higher ligation efficiency for A/T overhangs than for C/G overhangs.Similar to T4 DNA Ligase, the addition of PEG 6000 to the T3 DNA Ligase reaction mixture significantly improves its ligation efficiency for blunt-ended double-stranded DNA; in the absence of PEG 6000, the ligation efficiency for blunt-ended DNA is relatively low. In addition, T3 DNA Ligase shows twice the NaCl tolerance of T4 DNA Ligase and retains more than 95% of its activity in the presence of 1.0 M NaCl or KCl.
| Source | Recombinant expressed in Escherichia coli
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| Appearance | Sterile liquid
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| Storage Buffer | 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH 7.4 @25℃) |
| Enzyme Concentration | 3 KU/μl |
| Purity | Free of other DNA ligases except T3 DNA Ligase. Free of endonucleases, exonucleases, RNases and phosphatases.
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| Activity Definition | One unit is defined as the amount of enzyme required to give 50% ligation of 100ng HindIII fragments of λ DNA in a total reaction volume of 20μl in 1 minute at 25℃ in 1× T3 DNA Ligase Reaction Buffer. |
Component List
T750867
| Component | 150KU | 600KU | 5×600KU
| Storage |
| T750867A | T3 DNA Ligase (3 KU/μl) | 50µl | 200µl | 5×200µl | -20℃. Avoid freeze/ Thaw cycle. |
| T750867B | 2× Reaction Buffer | 0.5ml | 2ml | 5×2ml | -20℃. Avoid freeze/ Thaw cycle. |
Product Applications
Free of other DNA ligases except T3 DNA Ligase. Free of endonucleases, exonucleases, RNases and phosphatases.
Product Advantages
T3 DNA Ligase shows twice the NaCl tolerance of T4 DNA Ligase and retains more than 95% of its activity in the presence of 1.0 M NaCl or KCl. Therefore, T3 DNA Ligase is an ideal choice for ligation experiments under high ionic strength conditions.
Instructions for Use
1.Prepare the reaction mixture on ice according to the table below (using a 20 μl system as an example):
| Reagent | Volume |
|---|
| 2× Reaction Buffer | 10 μl |
| Vector DNA | X μl（0.020 pmol） |
| Insert DNA | Y μl（0.060 pmol） |
| Ultrapure Water | (9−X−Y) μl |
| T3 DNA Ligase（3KU/μl） | 1 μl |
| Total Volume | 20 μl |
Note 1: It is recommended to perform the ligation reaction at a molar ratio of DNA fragment to linearized vector of 3:1.
Note 2: T3 DNA Ligase is recommended to be added last.
2. Mix thoroughly by pipetting up and down, and centrifuge briefly to collect any liquid adhering to the tube wall to the bottom.
3. Reaction conditions: Incubate at 25°C (or room temperature) for 15–30 minutes.
4. Immediately place the ligation product on ice after the reaction. Transfer approximately 5 μl of the ligation product into 50 μl of competent cells for transformation. The remaining sample can be stored at -20°C optionally.
Note 1: Heat inactivation is not recommended, as it will significantly reduce the transformation efficiency of the ligation product.
Note 2: To check ligation efficiency, the reaction product can also be analyzed by agarose gel or polyacrylamide gel electrophoresis, followed by imaging and analysis. If DNA needs to be recovered from an agarose gel, a DNA gel extraction kit is recommended.
Frequently Asked Questions
1. Can T3 DNA Ligase be used with a buffer that does not contain PEG 6000?
Yes. If PEG 6000 cannot be included in the reaction system, it is recommended to prepare a 2X Reaction Buffer without PEG 6000.
2. What potential factors may cause transformation failure when using T3 DNA Ligase for ligation?
The following factors may lead to ligation or transformation failure:
a. Lack of ATP or Mg²⁺ in the reaction mixture. ATP in the buffer may degrade gradually during long-term storage. It is recommended to use fresh buffer or supplement ATP to ensure ligation efficiency.
b. Presence of high salt or EDTA in the reaction system. Purification of ligation substrates is recommended to remove inhibitors.
c. Incomplete inactivation of phosphatases such as CIP, BAP or SAP during dephosphorylation. Complete removal or inactivation of phosphatase is recommended following the recommended procedure.
d. Excessively high DNA concentration in the ligation mixture, leading to linear DNA formation only. The total DNA concentration is recommended to be within 1–10 μg/mL.
e. Too much ligation mixture added to competent cells. It is recommended to add 1–5 μL of ligation product to 50 μL of competent cells.
f. Prolonged ligation in the presence of PEG 6000 may generate large DNA fragments that inhibit transformation and reduce efficiency.
g. Lack of purification of ligation products before electroporation. Salt and PEG 6000 in the buffer inhibit electroporation. Purification using a spin column is recommended.h. Incomplete digestion of the empty vector, resulting in mostly empty clones without the desired insert.
3. What other factors should be considered when troubleshooting low transformation efficiency?
a. Inviable or low-efficiency competent cells. Use fresh competent cells.
b. Presence of inverted or tandem repeat sequences toxic to E. coli in the ligated DNA.
c. Insert DNA from mammalian or plant sources may contain methylated cytosine degraded by many E. coli strains. Use mcrA, mcrBC, and mrr deficient strains.
d. Large vector size (>10 kb) is not suitable for chemical transformation; electroporation is recommended.
4. What issues during restriction enzyme digestion can cause ligation or subsequent transformation failure?
a. Low restriction efficiency or incomplete digestion. Ensure sufficient protection bases at the ends of PCR fragments (at least 6 extra bases outside the restriction site). Test enzyme activity using a control substrate.
b. Incomplete inactivation of restriction enzymes. Purify DNA if the enzyme cannot be heat-inactivated.
c. Star activity during digestion. Analyze DNA by gel electrophoresis; reduce enzyme amount or digestion time.
d. Contamination with exonucleases or phosphatases that damage DNA ends. Purify DNA.
5. How much DNA should be used with T3 DNA Ligase?
To promote circularization and improve transformation efficiency, the total DNA concentration should be 1–10 μg/mL.A molar ratio of insert to linearized vector of 3:1 is recommended.A ratio below 2:1 reduces ligation efficiency; above 6:1 may cause multiple inserts.If DNA concentration is unknown, test multiple ratios.
6. Does T3 DNA Ligase have higher salt tolerance than T4 DNA Ligase?
Yes. T3 DNA Ligase tolerates 250–300 mM salt in ligation reactions.Note that in the presence of PEG, DNA may precipitate at high salt and inhibit ligation.
7. Can T3 DNA Ligase be heat-inactivated?
Yes.In a PEG-free system, heat inactivation can be achieved at 65 °C for 10 minutes.In a PEG-containing system, heat inactivation is possible but not recommended for downstream transformation, as it reduces efficiency.
8. What controls should be used to test cells and DNA with T3 DNA Ligase?
Use the same DNA concentration for all controls, typically 0.1–1.0 ng per transformation.
a. Transform uncut vector into competent cells and plate on medium with/without antibiotic to test cell viability and antibiotic resistance.
b. Transform linearized vector to check undigested plasmid background. Colony number should be < 10% of control a.
c. Transform cut and re-ligated vector to test ligase activity and end integrity. Colony number should be close to control a.
d. Transform cut, dephosphorylated and re-ligated vector to check phosphatase background. Colony number should be < 10% of control a.
Notes
1. ATP is an essential cofactor for T3 DNA Ligase, unlike E. coli DNA Ligase which uses NAD⁺. A final ATP concentration of 1 mM is required. The supplied 2X Reaction Buffer contains ATP and PEG 6000.
2. T3 DNA Ligase acts on double-stranded DNA or nicked duplexes with a complete DNA strand. It does not ligate single-stranded DNA or RNA directly, but supports DNA-templated ligation of ssDNA, ssRNA, or ssDNA–ssRNA.
3. The standard reaction buffer contains 7.5% PEG 6000. If PEG is incompatible, prepare a PEG-free buffer or use T4 DNA Ligase buffer supplemented with 1 mM ATP; however, activity will decrease ~10-fold.
4. T3 DNA Ligase can be heat-inactivated only in PEG-free buffer. Heat inactivation in PEG-containing buffer severely reduces transformation efficiency.
5. If high NaCl is required, use a PEG-free buffer.
6. In a standard 20 μL vector–insert ligation, incubate at 25 °C for 30 minutes.
7. Use ultrapure water (DNase/RNase-free, sterile).
8. This product is for research use only by qualified personnel. Not for clinical diagnosis, therapy, food, or drug use. Do not store in residential areas.
9. For safety and health, wear lab coat and disposable gloves.
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