阿拉丁生产的 T4 DNA 连接酶可以催化粘端或平端双链 DNA 或 RNA 的 5'-P 末端和 3'-OH 末端之间以磷酸二酯键结合，该催化反应需 ATP 作为辅助因子。同时 T4 DNA 连接酶可以修补双链 DNA、双链 RNA 或 DNA/RNA 杂合物上的单链缺刻（single-strand nicks）。
| 来源（Source） | 大肠杆菌重组表达 |
| 外观（Appearance） | 无菌液体 |
| 保存液（Storage Buffer） | 20 mM Tris, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% (v/v) Glycerol, pH 7.5.
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| 酶浓度（Enzyme Concentration） | 1000 U/μl |
| 纯度（Purity） | 不含 DNA 内切酶、外切酶和磷酸酯酶，不含 RNA 酶，满足常规连接反应要求。
|
| 活性定义（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). 200U 等于 1 个 Weiss unit，以 Weiss unit 计，本产品共 200 单位。 |
组分表
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. |
产品应用
T4 DNA Ligase 常用于 DNA 片段和载体、linker 或 adaptor 等的连接。也可以用于缺刻修复及 Ligase 介导的 RNA 检测。
产品优势
修复及 Ligase 介导的 RNA 检测。
使用说明
1. PCR 产物或酶切片段和普通载体的连接：
a. 取 1-2μg 载体酶切过夜，或至少酶切 3-5 小时以上。尽量确保酶切充分，否则后续会导致产生很多自连的克隆。
b. 载体酶切完毕后，可以使用试剂盒进行纯化，例如 PCR 纯化试剂盒/DNA 纯化试剂盒。也可以采用常规的酚氯仿抽提乙醇沉淀方法纯化载体。对于酶切产生较大片段(大于 50-60bp)的情况推荐采用切胶回收的方式。
c. 对于 PCR 产物：PCR 产物凝胶电泳后，切胶回收预期大小的 DNA 片段。凝胶中 DNA 片段的回收可以使用试剂盒进行操作，也可以采用反复冻融等方法回收 DNA 片段。
d. 对于回收的 PCR 产物或其它需酶切的质粒或 DNA 片段，用适当内切酶酶切，随后纯化酶切产物。
注：这一步的酶切不必酶切特别充分，通常酶切效率能达到 80-90%以上即可。即本步骤的酶切通常酶切 1-2 小时即可。酶切产物可以使用试剂盒进行纯化，例如,PCR 纯化试剂盒/DNA 纯化试剂盒。也可以采用常规的酚氯仿抽提乙醇沉淀方法纯化。
e. 取约 25-100ng 经过酶切和纯化的载体，加入 3 倍摩尔数的待插入片段。参考下表设置连接反应体系。
注 1：很多时候由于载体量和待插入片段的量都比较少，在回收后很难定量。此时可根据回收前电泳条带的亮度进行大致的估计。通常以 DNA 分子量标准的某一条带为参考，估计或通过灰度半定量您的目的条带和该参考条带的亮度的比例关系。然后再按照预计的纯化或凝胶回收时的得率计算出最终得到的载体量和待插入片段量的比例关系。
注 2：通常每个反应使用 0.2-0.5μl 连接酶已经足够，如果希望进一步提高连接效率，可以把连接酶的用量提高至 1μl。
| 载体 | 约 50–100 ng |
| 待插入片段 | 约载体摩尔数的 3 倍 |
| 10× Ligation Buffer | 2 μl |
| 双蒸水或 Milli-Q 水 | 加至 20 μl |
| T4 DNA Ligase | 0.2–0.5 μl |
| 总体系 | 20 μl |
f. 用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。
g.20-25℃孵育连接 1-2 小时，或 16℃孵育连接过夜。
注 1：对于双平端连接必须连接过夜。对于双平端连接，直接使用 T4 DNA Ligase 连接效率较低，推荐使用快速 DNA 连接试剂盒。
注 2：为快速获得预期克隆可以参考如下方法：对于 20μl 的粘端连接反应，在连接 1-2 小时后可以取 10μl 直接转化大肠杆菌，其余 10μl 可以 16℃孵育连接过夜。如果第二天顺利获得克隆，即可进入下一步实验；如果第二天转化的大肠杆菌未获得预期的克隆，则可以取连接过夜的剩余连接产物再次转化大肠杆菌。
h. 随后即可直接取连接产物用于转化感受态细菌。
2. PCR 产物和 T 载体的连接：
a. PCR 产物凝胶电泳后，切胶回收预期大小的 DNA 片段。凝胶中 DNA 片段的回收可以使用试剂盒进行操作，也可以采用反复冻融等方法回收 DNA 片段。
b. 按照 T 载体的说明书取适量 T 载体，加入 3 倍摩尔数的待插入片段。参考下表设置连接反应体系。
注 1：很多时候由于载体量和 PCR 产物的量都比较少，在回收后很难定量。此时可以根据回收前电泳条带的亮度进行大致的估计。通常 以 DNA 分子量标准的某一条带为参考，估计或通过灰度半定量您的目的条带和该参考条带的亮度的比例关系。然后再按照预计的纯化或凝胶回收时的得率计算出最终得到的载体量和 PCR 产物的量的比例关系。
注 2：通常每个反应使用 0.2-0.5μl 连接酶已经足够，如果希望进一步提高连接效率，可以把连接酶的用量提高至 1μl。
| T 载体 | 适量 |
| 待插入片段 | 约载体摩尔数的 3 倍 |
| 10× Ligation Buffer | 2 μl |
| 双蒸水或 Milli-Q 水 | 加至 20 μl |
| T4 DNA Ligase | 0.2–0.5 μl |
| 总体系 | 约 20 μl |
c. 用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。
d.20-25℃孵育连接 1-2 小时，或 16℃孵育连接过夜。 注：为快速获得预期克隆可以参考如下方法：对于 20μl 的粘端连接反应，在连接 1-2 小时后可以取 10μl 直接转化大肠杆菌，其余 10μl 可以 16℃孵育连接过夜。如果第二天顺利获得克隆，即可进入下一步实验；如果第二天转化的大肠杆菌未获得预期的克隆，则可以取连接过夜的剩余连接产物再次转化大肠杆菌。
e. 随后即可直接取连接产物用于转化感受态细菌。
3. Linker 或 RNAi 片段和载体的连接：
a. 载体的酶切和纯化同步骤 1a 和 1b。
b. Linker 或 RNAi 片段的退火可以选择适当的 DNA 退火缓冲液，例如 Annealing Buffer for DNA Oligos(5X)， 进行退火反应。
c. 长度大于 8bp 的 Linker 或退火的 RNAi 片段，可以按照 5:1 至 10:1 的比例和载体进行连接反应。例如载体为 0.03pmol，则插入片 段可以为 0.15 至 0.3pmol。长度小于 8bp 的 linker，比例需调整为 10:1 以上。
d. 除插入片段的用量外，随后按照步骤 1e-1h 进行。
4. DNA 自身环化的连接：
参考步骤 1e，待插入片段换成适量的水即可。其余步骤按照步骤 1f-1h 进行。
5. 其它类型的 DNA 片段连接参考上述方法进行。
常见问题
1. 连接反应后转化效率很低或阳性克隆非常少：
a.可能感受态细菌转化效率太低，用质粒作为阳性对照同时检测感受态的转化效率。
b. 可以尝试提高载体或插入片段的纯度。对于平端连接需注意适当延长连接时间。
c. 可能载体酶切不够充分，用未经连接的载体转化作为阴性对照。
d. 用存放 DNA 的溶液进行转化，作为阴性对照，检测感受态细菌是否存在问题。
注意事项：
（1）对于普通的转化大肠杆菌的操作，不必对连接产物进行纯化，连接产物可以直接用于转化。但用电转方法转化大肠杆菌时，通常宜先用 DNA 纯化试剂盒或酚氯仿抽提方法等纯化 DNA，然后再进行电转。
（2）需进行平端连接或快速连接时，推荐使用快速 DNA 连接试剂盒。T4 DNA Ligase 可以进行平端连接，但效率较低。
（3）普通连接反应不必进行凝胶电泳观察。如果需要对于连接产物进行凝胶电泳观察，推荐先在 65℃孵育 10 分钟使。
（4）T4 DNA Ligase 失活，以避免 T4 DNA Ligase 和 DNA 结合导致的条带位置迁移(band shift)。
（5）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。
（6）为了您的安全和健康，请穿实验服并戴一次性手套操作。
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
|
| Appearance | Sterile liquid
|
| Storage Buffer | 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% (v/v) Glycerol.
|
| Enzyme Concentration | 1000 U/μl |
| Purity | Free of DNA endonucleases, exonucleases, phosphatases, and RNases, meeting the requirements for routine ligation reactions.
|
| 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.
ZJ26F0434238
危险品化学品经营许可证（带存储）