阿拉丁生产的T7 DNA Ligase，即T7 DNA连接酶，是由纯化获得的一种来源于T7噬菌体的、ATP依赖的双链DNA连接酶，并且对于粘性末端的连接效率远远高于平末端。与T3和T4 DNA Ligase不同，T7 DNA Ligase仅可催化双链DNA相邻粘性末端5'磷酸和3'羟基磷酸二酯键的形成，但不能有效催化平末端双链DNA的连接，加入大于或等于20%的PEG6000可以适当提高该酶的平末端DNA连接活性。因此在平末端和粘性末端双链DNA底物同时存在，且仅需连接粘性末端双链DNA的分子生物学实验中，T7 DNA Ligase是理想的选择。
| 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 T7 DNA Ligase 之外的其它种类的 DNA 连接酶，不含 DNA 内切酶和外切酶，不含 RNA 酶，不含磷酸酯酶。 | | 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 20 μl in 30 minutes at 25℃ in 1× T7 DNA Ligase Reaction Buffer. |
|
组分表
| T750877 | Component | 150KU | 600KU | 5×600KU | Storage | | T750877A | T7 DNA Ligase (3 KU/μl) | 50µl | 200µl | 5×200µl | -20℃. Avoid freeze/ Thaw cycle. | | T750877B | 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，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、定点突变和 Transcription Activator-Like Effector Nucleases(TALEN)的 DNA 片段的 Golden Gate Assembly、粘端特异性连接等。
产品优势
与 T3 和 T4 DNA Ligase 不同，T7 DNA Ligase 仅可催化双链 DNA 相邻粘性末端 5'磷酸和 3'羟基磷酸二酯键的形成，但不能有效催化平末端双链 DNA 的连接，加入大于或等于 20%的 PEG6000 可以适当提高该酶的平末端 DNA 连接活性。因此在平末端和粘性末端双链 DNA 底物同时存在，且仅需连接 粘性末端双链 DNA 的分子生物学实验中，T7 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 | | T7 DNA Ligase（3KU/μl） | 1 μl | | Total Volume | 20 μl |
|
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：T7 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.T7 DNA Ligase 只能催化粘性末端双链 DNA 分子的连接。那么 T7 DNA Ligase 可以连接多少长度的粘性末端?
T7 DNA Ligase 可以有效催化 2bp 或更长粘性末端的连接，不能连接 1bp 的粘性末端。通常情况下，T7 DNA Ligase 一般不能连接平末端双链 DNA； 但当反应体系中含有非常高浓度的 PEG6000 (20-30% w/v)时，T7 DNA Ligase 对平末端双链 DNA 也具有一定的连接活性。
2.T7 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，我们推荐自行配制仅去除了 PEG6000 的 2× Reaction Buffer。
3.使用 T7 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，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。
4.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。
5.在限制性内切酶消化过程中存在哪些问题会导致 T7 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。
6.使用 T7 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。
注意事项
(1) ATP 是 T7 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。
(2) T7 DNA Ligase 不能有效催化平末端双链 DNA 片段的连接，对于平末端双链 DNA 的连接建议使用 T4 DNA Ligase。
(3) T7 DNA Ligase 的催化底物是双链 DNA，不能用于单链 DNA 或 RNA 的连接反应。
(4) T7 DNA Ligase 的反应体系中含有 7.5% PEG6000。如果实验体系中不能加入 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，但 T7 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。
(5) 反应体系所需的超纯水推荐使用 Ultrapure Water (DNase/RNase-Free, Sterile)。
(6) 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。
(7) 为了您的安全和健康，请穿实验服并戴一次性手套操作。
T7 DNA Ligase produced by Aladdin is an ATP-dependent double-stranded DNA ligase derived from T7 bacteriophage and obtained by purification. Its ligation efficiency for sticky ends is much higher than that for blunt ends. Unlike T3 and T4 DNA Ligase, T7 DNA Ligase can only catalyze the formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl groups of adjacent sticky ends on double-stranded DNA, but cannot efficiently ligate blunt-ended double-stranded DNA. The addition of PEG 6000 at a concentration of ≥20% can moderately improve the blunt-end DNA ligation activity of this enzyme. Therefore, T7 DNA Ligase is an ideal choice in molecular biology experiments where both blunt-ended and sticky-ended double-stranded DNA substrates are present and only sticky-ended double-stranded DNA ligation is required.
| Source | Recombinant expressed in Escherichia coli
| | Appearance | Sterile liquid
| | 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 from other DNA ligases except T7 DNA Ligase, free from DNA endonucleases and exonucleases, free from RNases, and free from phosphatases.
| | 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 20 μl in 30 minutes at 25℃ in 1× T7 DNA Ligase Reaction Buffer. |
|
Component List
| T750877 | Component | 150KU | 600KU | 5×600KU
| Storage | | T750877A | T7 DNA Ligase (3 KU/μl) | 50µl | 200µl | 5×200µl | -20℃. Avoid freeze/ Thaw cycle. | | T750877B | 2× Reaction Buffer | 0.5ml | 2ml | 5×2ml | -20℃. Avoid freeze/ Thaw cycle. |
|
Product Applications
Cloning of restriction enzyme-digested DNA fragments, ligation of double-stranded DNA to adapters, circularization of linear double-stranded DNA, nick repair of double-stranded DNA, site-directed mutagenesis, Golden Gate Assembly of DNA fragments for Transcription Activator-Like Effector Nucleases (TALEN), sticky end-specific ligation, etc.
Product Advantages
Unlike T3 and T4 DNA Ligase, T7 DNA Ligase can only catalyze the formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl groups of adjacent sticky ends on double-stranded DNA, but cannot efficiently ligate blunt-ended double-stranded DNA. The addition of PEG 6000 at a concentration of ≥20% can moderately improve the blunt-end DNA ligation activity of this enzyme. Therefore, T7 DNA Ligase is an ideal choice in molecular biology experiments where both blunt-ended and sticky-ended double-stranded DNA substrates are present and only sticky-ended double-stranded DNA ligation is required.
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 | | T7 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: T7 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. T7 DNA Ligase only catalyzes the ligation of sticky-ended double-stranded DNA molecules. What length of sticky ends can T7 DNA Ligase ligate?
T7 DNA Ligase can efficiently catalyze the ligation of sticky ends of 2 bp or longer, but cannot ligate 1 bp sticky ends. Under normal conditions, T7 DNA Ligase generally cannot ligate blunt-ended double-stranded DNA; however, when the reaction system contains a very high concentration of PEG 6000 (20–30% w/v), T7 DNA Ligase also exhibits certain blunt-end ligation activity.
3. Can T7 DNA Ligase be used with a buffer that does not contain PEG 6000?
Yes. If PEG 6000 cannot be added to the experimental system, we recommend preparing a 2× Reaction Buffer without PEG 6000.
3. What potential factors can lead to transformation failure when performing ligation with T7 DNA Ligase?
The following factors can cause ligation failure:
a. Lack of ATP or Mg²⁺ in the reaction system leads to ligation failure. ATP in the buffer may degrade gradually over long-term storage, causing this issue. It is recommended to use freshly provided buffer or supplement an appropriate amount of ATP to the reaction system to ensure ligation efficiency.
b. High salt or EDTA in the reaction system leads to ligation failure. It is recommended to purify the ligation substrates to remove interfering substances.
c. Phosphatases such as CIP, BAP, or SAP are not completely inactivated during dephosphorylation. It is recommended to completely remove the phosphatase following the recommended procedure.
d. Excessively high DNA concentration in the reaction system results in only linear DNA formation. It is recommended to maintain the total DNA concentration in the ligation system within the range of 1–10 μg/ml.
e. Adding too much ligation product to competent cells causes transformation failure. 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 gradually produces large DNA fragments that inhibit transformation, reducing efficiency.
g. The ligation product is not purified before electroporation. Salt and PEG 6000 present in the buffer inhibit electroporation. It is recommended to purify the ligation product using a purification column to remove the buffer as much as possible.
h. Incomplete digestion of the empty vector results in most clones being empty vectors lacking the desired insert-containing clones.
4. What other factors should be considered when troubleshooting transformation efficiency?
a. Incompetent or low-efficiency competent cells. It is recommended to use fresh competent cells.
b. The ligated DNA contains inverted or tandem repeats toxic to Escherichia coli.
c. Inserted DNA fragments from mammals or plants may contain methylated cytosines that can be degraded by many E. coli strains. It is recommended to use E. coli strains deficient in mcrA, mcrBC, and mrr.
d. The constructed vector is too large (>10 kb) and cannot be transformed chemically; electroporation is recommended.
5. What issues during restriction enzyme digestion can lead to failure in T7 DNA Ligase ligation or subsequent transformation?
a. Low digestion efficiency with incomplete cleavage. If cleavage occurs at the end of a PCR fragment, ensure sufficient protection bases are present; it is recommended to add an additional 6 bases outside the restriction site. It is also recommended to test the restriction enzyme activity using a control substrate.
b. Incomplete inactivation of the restriction enzyme. If the restriction enzyme cannot be heat-inactivated, purify the DNA to remove the enzyme as much as possible.
c. Star activity occurs when the restriction enzyme cuts the DNA fragment or vector. It is recommended to check the DNA by gel electrophoresis, reduce the amount of restriction enzyme used, or shorten the digestion time.
d. Presence of exonucleases or phosphatases in the DNA or restriction enzyme that damage DNA fragment ends; DNA purification is recommended.
6. How much DNA should be added when using T7 DNA Ligase?
To promote the formation of circular DNA ligation products and improve transformation efficiency, the total DNA concentration added should be between 1–10 μg/ml for efficient ligation. It is also recommended to add the insert DNA fragment and linearized vector at a molar ratio of 3:1. A molar ratio below 2:1 reduces ligation efficiency; a ratio above 6:1 may lead to multiple fragment insertions. If the substrate DNA concentration cannot be determined, multiple ratios can be tested.
Notes
(1) ATP is an essential cofactor for T7 DNA Ligase catalytic activity, unlike E. coli DNA Ligase, which uses NAD⁺ as a cofactor.
(2) T7 DNA Ligase cannot efficiently catalyze the ligation of blunt-ended double-stranded DNA fragments. For blunt-end ligation, T4 DNA Ligase is recommended.
(3) T7 DNA Ligase acts on double-stranded DNA and cannot be used for ligation of single-stranded DNA or RNA.
(4) The reaction system for T7 DNA Ligase contains 7.5% PEG 6000. If PEG 6000 cannot be added to the experimental system, consider preparing a ligation buffer without PEG 6000 or using the T4 DNA Ligase buffer system; however, T7 DNA Ligase activity is reduced by approximately 10-fold in the T4 DNA Ligase buffer system.
(5) Ultrapure Water (DNase/RNase-Free, Sterile) is recommended for the reaction system.
(6) This product is limited to scientific research use by professionals only. It must not be used for clinical diagnosis or treatment, food or drug applications, or stored in ordinary residential premises.
ZJ26F0434251
危险品化学品经营许可证（带存储）