T4 DNA Ligase produced by Aladdin catalyzes the formation of a phosphodiester bond between the 5'-phosphoryl end and the 3'-hydroxyl end of double-stranded DNA or RNA with cohesive or blunt ends. This catalytic reaction requires ATP as a cofactor. Meanwhile, T4 DNA Ligase can repair single-strand nicks in double-stranded DNA, double-stranded RNA, or DNA/RNA hybrids.
| Source | Recombinant expressed in Escherichia coli
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| Appearance | Sterile liquid
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| Storage Buffer | 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% (v/v) Glycerol.
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| Enzyme Concentration | 1000 U/μl |
| Purity | Free of DNA endonucleases, exonucleases, phosphatases, and RNases, meeting the requirements for routine ligation reactions.
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| Activity Definition | One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 min at 16℃ in 20 μl of the assay mixture containing 50 mM Tris, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA and a 5'-DNA termini concentration of 0.12 μM (300 μg/ml). 200 U is equivalent to 1 Weiss unit. The total activity of this product is 200 units, expressed in Weiss units. |
Component List
T665705
| Component | 40KU
| 5×40KU | Storage |
| T665705A | T4 DNA Ligase (1000U/μl)
| 40µl | 5×40µl | -20℃. Avoid freeze/ Thaw cycle. |
| T665705B | 10× Ligation Buffer | 300µl
| 5×300µl | -20℃. Avoid freeze/ Thaw cycle. |
Product Applications
T4 DNA Ligase is commonly used for the ligation of DNA fragments to vectors, linkers, adaptors, etc. It can also be used for nick repair and ligase-mediated RNA detection.
Product Advantages
Nick repair and ligase-mediated RNA detection.
Instructions for Use
1. Ligation of PCR products or digested fragments with conventional vectors:
a. Digest 1–2 μg of vector overnight, or for at least 3–5 hours. Ensure complete digestion as far as possible; otherwise, many self-ligated colonies will be generated in subsequent steps.
b. After vector digestion, purify using a purification kit, such as a PCR purification kit / DNA purification kit. Conventional phenol-chloroform extraction followed by ethanol precipitation can also be used. For large fragments (>50–60 bp) generated by digestion, gel extraction is recommended.
c. For PCR products: After gel electrophoresis of PCR products, excise and recover the DNA fragment of the expected size. Recovery from the gel can be performed using a kit or by repeated freeze-thaw methods.
d. Digest the recovered PCR product or other plasmid/DNA fragments requiring digestion with an appropriate restriction enzyme, then purify the digested product.
Note: Digestion at this step does not need to be highly complete; an efficiency of over 80–90% is generally sufficient. Digestion for 1–2 hours is usually adequate. The digested product can be purified using a kit (e.g., PCR purification kit / DNA purification kit) or by conventional phenol-chloroform extraction and ethanol precipitation.
e. Use approximately 25–100 ng of digested and purified vector, and add insert DNA at a 3:1 molar ratio relative to the vector. Set up the ligation reaction according to the table below.
Note 1: In many cases, the amounts of vector and insert are too low to quantify accurately after recovery. In such cases, estimate based on the brightness of electrophoresis bands before recovery. Use a band from the DNA molecular weight marker as a reference, estimate or semi-quantify the brightness ratio between your target band and the reference band, then calculate the final ratio of vector to insert based on the expected recovery yield from purification or gel extraction.
Note 2: 0.2–0.5 μL of ligase per reaction is usually sufficient. The ligase volume can be increased to 1 μL to further improve ligation efficiency.
| Vector | approx. 50–100 ng |
| Insert fragment | approx. 3× molar amount relative to vector |
| 10× Ligation Buffer | 2 μl |
| Double-distilled water or Milli-Q water | add to 20 μl |
| T4 DNA Ligase | 0.2–0.5 μl |
| Total volume | 20 μl |
f. Mix gently by pipetting up and down or brief vortexing, then centrifuge at room temperature for several seconds to collect the liquid at the bottom of the tube.
g. Incubate at 20–25°C for 1–2 hours for ligation, or at 16°C overnight.
Note 1: For blunt‑blunt end ligation, overnight incubation is mandatory. Direct ligation with T4 DNA Ligase shows low efficiency for blunt‑blunt ends; a rapid DNA ligation kit is recommended.
Note 2: For rapid clone acquisition: in a 20 μL cohesive‑end ligation, 10 μL can be used directly for E. coli transformation after 1–2 hours of ligation, and the remaining 10 μL can be incubated at 16°C overnight. If colonies are obtained the next day, proceed to the next step; if not, use the remaining overnight‑ligated product for a second transformation.
h. The ligation product can then be used directly for transformation of competent cells.
2. Ligation of PCR products with T‑vectors:
a. After gel electrophoresis of the PCR product, excise and recover the DNA fragment of the expected size. Fragment recovery from the gel can be performed using a kit or by repeated freeze‑thaw methods.
b. Use an appropriate amount of T‑vector according to the T‑vector manual, and add insert fragment at a 3:1 molar ratio. Set up the ligation reaction according to the table below.
Note 1: In many cases, vector and PCR product amounts are too low to quantify accurately after recovery. Estimate based on band brightness before recovery, using a DNA marker band as reference. Estimate or semi‑quantify the brightness ratio between the target band and the reference band, then calculate the final vector‑to‑insert ratio based on the expected purification or gel recovery yield.
Note 2: 0.2–0.5 μL of ligase per reaction is usually sufficient. The ligase volume can be increased to 1 μL to further improve ligation efficiency.
| T-vector | as appropriate |
| Insert fragment | approx. 3× molar amount relative to vector |
| 10× Ligation Buffer | 2 μl |
| Double-distilled water or Milli-Q water | add to 20 μl |
| T4 DNA Ligase | 0.2–0.5 μl |
| Total volume | approx. 20 μl |
c. Mix gently by pipetting up and down or brief vortexing, then centrifuge at room temperature for several seconds to collect the liquid at the bottom of the tube.
d. Incubate at 20–25°C for 1–2 hours for ligation, or at 16°C overnight.
Note: For rapid acquisition of desired clones: In a 20 μL cohesive-end ligation reaction, 10 μL can be used directly for E. coli transformation after 1–2 hours of ligation, and the remaining 10 μL can be incubated at 16°C overnight. If colonies are obtained the next day, proceed to the next step; if not, use the remaining overnight-ligated product for a second transformation.
e. The ligation product can then be used directly for transformation of competent bacteria.
3. Ligation of Linker or RNAi fragments with vectors:
a. Digestion and purification of the vector are the same as steps 1a and 1b.
b. Annealing of Linker or RNAi fragments can be performed using an appropriate DNA annealing buffer, such as Annealing Buffer for DNA Oligos (5X).
c. For Linkers or annealed RNAi fragments longer than 8 bp, ligation with the vector can be performed at a molar ratio of 5:1 to 10:1 (insert:vector). For example, if the vector is 0.03 pmol, the insert can be 0.15 to 0.3 pmol. For linkers shorter than 8 bp, the ratio should be adjusted to 10:1 or higher.
d. Except for the amount of insert fragment, proceed with steps 1e–1h.
4. Self-circularization of DNA:
Refer to step 1e, replacing the insert fragment with an appropriate volume of water. Follow steps 1f–1h for the remaining procedures.
5. Ligation of other types of DNA fragments can be performed with reference to the methods above.
FAQ
1. Very low transformation efficiency or very few positive colonies after ligation:
a. The transformation efficiency of competent bacteria may be too low; use a plasmid as a positive control to test competent cell efficiency.
b. Try improving the purity of the vector or insert. For blunt-end ligation, ensure appropriate extension of ligation time.
c. The vector may not be fully digested; use unligated vector for transformation as a negative control.
d. Use the solution storing DNA for transformation as a negative control to check for issues with competent bacteria.
Precautions
(1) For routine E. coli transformation, purification of the ligation product is unnecessary; the ligation mixture can be used directly for transformation. However, for electroporation of E. coli, it is generally recommended to purify the DNA first using a DNA purification kit or phenol-chloroform extraction before electroporation.
(2) For blunt-end ligation or rapid ligation, a rapid DNA ligation kit is recommended. T4 DNA Ligase can perform blunt-end ligation but with lower efficiency.
(3) Routine ligation reactions do not require gel electrophoresis analysis. If gel electrophoresis is needed, incubate at 65°C for 10 minutes first.
(4) Inactivate T4 DNA Ligase to avoid band shift caused by T4 DNA Ligase binding to DNA.
(5) This product is for scientific research use only by qualified personnel. It is not intended for clinical diagnosis or treatment, food or pharmaceutical applications, and must not be stored in ordinary residential premises.
(6) For your safety and health, please wear a lab coat and disposable gloves during operation.
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