阿拉丁生产的T3 DNA Ligase 即 T3 DNA 连接酶，是本公司技术平台表达、纯化获得的一种来源于 T3 噬菌体、ATP 依赖型的双链 DNA 连接酶。T3 DNA Ligase 能够有效催化粘性末端和平末端双链 DNA 分子的连接以及双链 DNA 或 DNA 与 RNA 杂合双链的缺刻修复。缺刻修复时，完整链为 DNA，缺刻链 5’端和 3’端’分别为 DNA-DNA、DNA-RNA、RNA-DNA 或 RNA-RNA 都是可以被 T3 DNA Ligase 修复的，因此 T3 DNA Ligase 也可以用于生成 DNA-RNA 和 RNA-DNA 融合链接的核酸，以及用于基于 DNA 模板的 RNA 连接以形成较长的 RNA 片段。T3 DNA Ligase 能够催化双链 DNA 中临近的 5'磷酸和 3' 羟基之间形成磷酸二酯键，且对 A/T 突出末端的连接效率高于 C/G 末端。与 T4 DNA Ligase 相同，在 T3 DNA Ligase 反应体系中加入 PEG6000 可以 显著提高其对平末端双链 DNA 的连接效率；而在缺少 PEG6000 的连接体系中，T3 DNA Ligase 对平末端双链 DNA 分子的连接效率较低。另外，T3 DNA Ligase 对 NaCl 的耐受性是 T4 DNA Ligase 的 2 倍，其在 1.0M NaCl 或 KCl 的条件下仍可保持 95%的活性。
| 来源（Source） | 大肠杆菌重组表达 |
| 外观（Appearance） | 无菌液体 |
| 保存液（Storage Buffer） | 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH 7.4 @25℃) |
| 酶浓度（Enzyme Concentration） | 3 KU/μl |
| 纯度（Purity） | 不含除 T3 DNA Ligase 之外的其它种类的 DNA 连接酶，不含内切酶和外切酶，不含 RNA 酶，不含磷酸酯酶。
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| 活性定义（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.
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组分表
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. |
产品应用
限制性内切酶切 DNA 片段的克隆，PCR 产物的克隆连接，双链 DNA 和接头的连接，线性双链 DNA 的环化，双链 DNA 的缺刻修复，定点突变，高盐体系的连接，双链 DNA 缺刻修复，DNA 引导的 DNA 与 RNA 连接，DNA 引导的 RNA 连接。
产品优势
T3 DNA Ligase 对 NaCl 的耐受性是 T4 DNA Ligase 的 2 倍，其在 1.0M NaCl 或 KCl 的条件下仍可保持 95%的活性。因此，在涉及到高离子浓度连接的实验中，T3 DNA Ligase 是理想的选择。
使用说明
1.参考下表在冰浴中配制反应体系(以 20μl 体系为例)：
| 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 |
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注 1：建议按照 DNA 片段与线性化载体摩尔比 3:1 的比例进行连接反应。
注 2：T3 DNA Ligase 建议最后添加。
2.移液器吹打混匀，低速离心使粘附在管壁上的液体沉降至管底。
3.反应条件：25℃(或室温)孵育 15~30 分钟。
4.反应完成后需将连接产物立即置于冰上，取 5μl 左右连接产物转化到 50μl 感受态细胞中。剩余样品可选择性的保存于-20℃。
注 1：不能进行热失活，热失活会显著降低连接产物的转化效率。
注 2：为检测连接效率，也可将反应后的产物进行琼脂糖凝胶或聚丙烯凝胶电泳，拍照观察并分析连接效果。如果需要从琼脂糖凝胶中回收 DNA 样品，推荐使用 DNA 凝胶回收试剂盒。
常见问题
1.T3 DNA Ligase 可以和不含 PEG6000 的缓冲液一起使用吗?
可以。如果实验体系中不能加入 PEG6000，推荐自行配制仅去除了 PEG6000 的 2X Reaction Buffer。
2.使用 T3 DNA Ligase 进行连接反应时，有哪些潜在的因素会导致转化失败?
有以下因素会导致连接反应失败：
a.反应体系中缺少 ATP 或 Mg²⁺会导致连接失败。缓冲液中的 ATP，保存时间过长可能会逐渐降解导致此问题的发生。建议使用新鲜提供的缓冲液或适量补充 ATP 到反应体系中，以保证连接效率。
b.反应体系中存在高盐或 EDTA 会导致连接失败。建议纯化连接底物，去除干扰。
c.CIP、BAP 或 SAP 等磷酸酶在去磷酸化过程中没有完全失活。建议按照推荐的步骤完全去除磷酸酶。
d.反应体系中 DNA 的浓度过高会导致只产生线性 DNA。建议连接体系中 DNA 的总浓度保持在 1-10μg/ml 的范围内。
e.加入太多的连接产物转化到感受态细胞中会导致转化的失败。建议加入 1-5μl 的连接产物转化到 50μl 的感受态细胞中。
f.含有 PEG6000 的情况下过长时间的连接，会逐渐产生抑制转化的大片段 DNA，会降低转化效率。
g.连接产物在电穿孔前没有进行纯化。缓冲液中存在的盐和 PEG6000 均会抑制电穿孔实验。建议使用纯化柱对连接产物进行纯化，以尽可能去除缓冲液。
h.空载体酶切不完全，会导致获得的克隆基本都是空载体而缺少含有插入片段的目的克隆。
3.在解决转化效率问题时还应考虑哪些因素?
a.感受态细胞不能存活或转化效率过低。建议使用新的感受态细胞。
b.连接的 DNA 是否含有大肠杆菌拮抗的反向重复序列或串联重复序列。
c.插入的 DNA 片段如果来自哺乳动物或者植物，可能含有能够被多种大肠杆菌株所降解的甲基化胞嘧啶。建议使用 mcrA、mcrBC 和 mrr 缺乏的大肠杆菌株。
d.构建的载体过大(>10kb)，不能使用化学转化的方式，建议使用电穿孔转化方式。
4.在限制性内切酶消化过程中存在哪些问题会导致 T3 DNA Ligase 的连接反应或后续的转化失败?
a.酶切效率不高，没有实现完全酶切。如果切割发生在一个 PCR 片段的末端，需要确保有足够的酶切保护碱基，建议在酶切位点外侧额外增加 6 个碱基。并建议使用对照底物测试限制酶的活性。
b.限制性内切酶没有完全失活。如果限制性内切酶不能热失活，可以纯化 DNA 以尽可能地去除限制性内切酶。
c.限制性内切酶切割 DNA 片段或载体时产生了星活性。建议凝胶电泳检测 DNA，适当减少限制性内切酶的使用量或减少酶切反应时间。
d.DNA 或限制性内切酶中含有破坏 DNA 片段末端的核酸外切酶或磷酸酶时，建议纯化 DNA。
5.使用 T3 DNA Ligase 时应加入多少 DNA?
为促进环化 DNA 连接产物的形成，提高转化效率，加入的总 DNA 浓度应在 1-10μg/ml 之间，以实现有效连接。同时建议按照插入 DNA 片段与线性化载体摩尔比 3:1 的比例加入到反应体系中。摩尔比低于 2:1 会降低连接效率；摩尔比高于 6:1 会导致多个片段的插入。如果底物 DNA 的浓度无法确定，可尝试多种比例的连接。
6.T3 DNA 连接酶对盐离子是否比 T4 DNA 连接酶具有更高的耐受性?
是的。连接反应中 T3 DNA Ligase 能够耐受 250-300mM 的盐浓度。同时请注意，在 PEG 存在的连接体系中，DNA 在高盐条件下会沉淀，并抑制连接反应的进行。
7.T3 DNA Ligase 能否被热失活?
可以。在不含有 PEG 的反应体系中 65℃孵育 10 分钟能够将 T3 DNA Ligase 热失活；但在含有 PEG 的反应体系中，T3 DNA Ligase 可以进行热失活，但会影响后续的转化效率。因此在后续用于转化的情况下，不建议进行热失活。
8.当使用 T3 DNA 连接酶时，应该使用什么对照来测试细胞和 DNA?
注：对每个对照均使用相同的 DNA 浓度，通常每次转化 0.1-1.0ng 的 DNA。
a.在感受态细胞中转化未经酶切线性化的载体并均匀涂布到不含有/含有抗生素的培养基平板上，可检测细胞活力和质粒的抗生素耐受性。
b.检测线性化后的载体是否含有未被完全切割的质粒背景。将线性化后的载体转化感受态细胞，获得的克隆数目应小于步骤 a 中克隆数目的 10%。
c.使用切割并重新连接的质粒检测连接酶的活性和 DNA 末端的完整性。将切割并修饰后的载体转化感受态细胞，获得的克隆数目应接近步骤 a 中克隆数目。
d.使用切割，磷酸化并重新连接的质粒检测由于磷酸化酶处理不完全所导致的背景。将切割，磷酸化并重新连接的质粒转化感受态细胞，获得的克隆数目应小于步骤 a 中克隆数目的 10%。
注意事项
(1) ATP 是 T3 DNA Ligase 发挥催化活性所必需的辅助因子，而不是像 E.coli DNA Ligase 以 NAD 作为辅助因子的。因此连接反应体系中一定要保持终浓度为 1mM 的 ATP。本产品提供的 2X Reaction Buffer 中已经包含了 ATP 和连接粘末端所需的 PEG6000。
(2) T3 DNA Ligase 的催化底物是双链 DNA 或完整链为 DNA 的缺刻双链，不能直接用于单链 DNA 或 RNA 的连接反应，但可以用于 DNA 模板引导的单链 DNA 之间、单链 RNA 之间或单链 DNA 与单链 RNA 之间的连接。
(3) T3 DNA Ligase 的反应体系中含有 7.5% PEG6000。如果后续实验体系不兼容 PEG6000，可考虑自行配制不含 PEG6000 的连接反应缓冲液，或者使用 T4 DNA Ligase 的连接缓冲体系，同时注意添加终浓度为 1mM 的 ATP，但 T3 DNA Ligase 连接酶在 T4 DNA Ligase 的连接缓冲体系中 的活性会降低约 10 倍。
(4) 在不含 PEG 的反应体系中，T3 DNA Ligase 可进行热失活。如果反应体系中含有 PEG6000，T3 DNA Ligase 不能进行热失活，否则会显著降低后续的转化效率。
(5) 如果连接体系中需要维持高浓度 NaCl，建议使用不含 PEG6000 的缓冲液。
(6) 在标准的载体与片段连接的 20μl 反应体系中，通常需要在 25℃下反应 30 分钟。
(7) 反应体系所需的超纯水推荐使用 Ultrapure Water (DNase/RNase-Free, Sterile) 。
(8) 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
(9) 为了您的安全和健康，请穿实验服并戴一次性手套操作。
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.
ZJ26F0434279
危险品化学品经营许可证（带存储）