T4 DNA Ligase 是从表达T4 DNA Ligase 基因的大肠杆菌经诱导表达后分离纯化而来的，催化相邻DNA链的5’磷酸基团和3’羟基基团以磷酸二酯键结合反应。该酶可催化平末端或粘性末端DNA的连接，修复双链DNA、RNA、DNA/RNA杂交中的单链中的单链切口，但是对于单链核苷酸，没有活性。 | T665705 | Component | 100 U | 500 U | 5 KU | Storage | | T665705A | T4 DNA Ligase, 5 U/μL | 20 μL | 100 μL | 1 mL | -20℃. Avoid freeze/thaw cycle. | | T665705B | 10×Ligation Buffer | 150 μL | 750 μL | 5×1.5 mL | -20℃. Avoid freeze/thaw cycle. | | T665705C | 50% PEG Solution | 150 μL | 750 μL | 5×1.5 mL | -20℃. Avoid freeze/thaw cycle. |
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实验前准备及重要注意事项：
1.T4 DNA Ligase的最终用量不要超过推荐的用量，否则影响连接效率。
2，PEG可以极大提高平末端的连接效率，我们推荐加入终浓度为5% PEG Solution以提
高平末端的连接效率。
3，为了提高转化效率，建议所加入连接产物的量不要超过感受态细胞体积的10%
4.由于T4 DNA Ligase中含有甘油，比较粘稠容易挂壁，建议使用之前短暂离心将液体
收集到管底，取样时枪头尽量不要深入液面太深以免粘在枪头上造成损失
使用方法：
i粘性末端的连接
1.反应体系:
| 组分 | 20 μL 反应体系 | 终浓度 | | 线性载体DNA | X μL | 20-100 ng | | 插入DNA片段 | Y μL | 插入片段:载体1:1-5:1 | | 10×Ligation Buffer | 2 μL | / | | T4 DNA Ligase 5 U/ μL | 0.2 μL | 1 U | | ddH20 | 补充至20 μL | 20 μL |
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2.涡旋震荡，瞬间离心，将管壁上的溶液收集到管底。
3.反应条件:22C孵育10分钟。
4.瞬间离心，将管壁上的溶液收集到管底，65C孵育10分钟或70C孵育5分钟以灭活T4 DNA Ligase
5.可取5pL连接产物热击转化5uL感受态细胞或取15pL连接产物电击转化50pL感受态细胞。
注意:如需电击转化，推荐用乙醇沉淀法去除T4 DMA Ligase后进行电击转化 ii 平末端的连接 1.反应体系： | 组分 | 20 μL 反应体系 | 终浓度 | | 线性载体DNA | X μL | 20-100 ng | | 插入DNA片段 | Y μL | 插入片段:载体1:1-5:1 | | 10xLigation Buffer | 2 μL | / | | T4 DNA Ligase，5 U/uL | 1 μL | 5 U | | 50%PEG Solution | 2 L | 5% | | ddH20 | 补充至20 μL | 20 μL |
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2.涡旋震荡，瞬间离心，将管壁上的溶液收集到管底。
3，反应条件:22C孵育1小时。
4.瞬间离心，将管壁上的溶波收集到管底，65C孵育10分钟或70C孵育5分钟以灭活T4 DNA Ligase
5.可取5L连接产物热击转化50pL感受态细胞或取1-2L连接产物电击转化50pL感受态细胞。
注意:如需电击转化，推荐用乙醇沉淀法去除T4 DNA Ligase后进行电击转化
iii 线性DNA的自身环化
1.反应体系: | 组分 | 50 μL 反应体系 | 终浓度 | | 线性载体DNA | X μL | 5-50 ng | | 10xLigation Buffer | 5 μL | / | | T4 DNA Ligase，5 U/uL | 1 μL | 5 U | | ddH20 | 补充至50 μL | 50 μL |
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2.涡旋震荡，瞬间离心，将管壁上的溶液收集到管底。
3，反应条件:粘性末端22C孵育10分钟:平末端22C孵育1小时。
4.瞬间离心，将管壁上的溶液收集到管底，65C孵育10分钟或70C孵育5分钟以灭活T4 DNA Ligase
5.可取5pL连接产物热击转化5pL感受态细胞或取1-2pL连接产物电击转化50pL感受态细胞。
注意:如需电击转化，推荐用乙醇沉淀法去除T4 DNA Ligase后进行电击转化
T4 DNA Ligase is isolated and purified from Escherichia coli expressing the T4 DNA Ligase gene after induction and expression. It catalyzes the binding reaction of adjacent DNA strands with 5 'phosphate groups and 3' hydroxyl groups via phosphodiester bonds. This enzyme can catalyze the connection of flat or sticky end DNA, repairing single stranded incisions in double stranded DNA, RNA, and DNA/RNA hybridization, but has no activity for single stranded nucleotides. | T665705 | Component | 100 U | 500 U | 5 KU | Storage | | T665705A | T4 DNA Ligase, 5 U/μL | 20 μL | 100 μL | 1 mL | -20℃. Avoid freeze/thaw cycle. | | T665705B | 10×Ligation Buffer | 150 μL | 750 μL | 5×1.5 mL | -20℃. Avoid freeze/thaw cycle. | | T665705C | 50% PEG Solution | 150 μL | 750 μL | 5×1.5 mL | -20℃. Avoid freeze/thaw cycle. |
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Preparation and important precautions before the experiment:
The final dosage of T4 DNA Ligase should not exceed the recommended dosage, otherwise it will affect the connection efficiency.
2. PEG can greatly improve the connection efficiency of the flat end. We recommend adding a final concentration of 5% PEG Solution to improve the connection efficiency
The connection efficiency of the high flat end.
3. To improve conversion efficiency, it is recommended that the amount of connecting products added should not exceed 10% of the volume of receptive cells
4. Due to the presence of glycerol in T4 DNA ligase, which is viscous and prone to wall sticking, it is recommended to briefly centrifuge the liquid before use
Collect to the bottom of the tube, and when sampling, try not to let the nozzle go too deep into the liquid level to avoid sticking to the nozzle and causing losses
Usage:
i Connection at the adhesive end
1. Reaction system: | Component | 20 μL Reaction system | Final concentration | | Linear vector DNA | X μL | 20-100 ng | | Inserting DNA fragments | Y μL | Insertion fragments: vector 1:1-5:1 | | 10×Ligation Buffer | 2 μL | / | | T4 DNA Ligase 5 U/ μL | 0.2 μL | 1 U | | ddH20 | Add to 20 μL | 20 μL |
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2. Vortex oscillation, instant centrifugation, collects the solution on the pipe wall to the bottom of the pipe.
3. Reaction conditions: Incubate at 22C for 10 minutes.
4. Instantly centrifuge and collect the solution on the tube wall to the bottom of the tube. Incubate at 65C for 10 minutes or 70C for 5 minutes to inactivateT4 DNA Ligase
5. Take 5pL of the connecting product and heat shock transform it into 5uL of competent cells, or take 15pL of the connecting product and shock transform it into 50pL of competent cells Cells.
Attention: If electric shock conversion is required, it is recommended to use ethanol precipitation method to remove T4 DMA Ligase before conducting electric shock conversion
Ii. Connection at the flat end
1. Reaction system: | Component | 20 μL Reaction system | Final concentration | | Linear vector DNA | X μL | 20-100 ng | | Inserting DNA fragments | Y μL | Insertion fragments: vector 1:1-5:1 | | 10xLigation Buffer | 2 μL | / | | T4 DNA Ligase，5 U/uL | 1 μL | 5 U | | 50%PEG Solution | 2 L | 5% | | ddH20 | Add to 20 μL | 20 μL |
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2. Vortex oscillation, instant centrifugation, collects the solution on the pipe wall to the bottom of the pipe.
3. Reaction conditions: Incubate at 22C for 1 hour.
4. Instantly centrifuge and collect the dissolved waves on the tube wall to the bottom of the tube. Incubate at 65C for 10 minutes or 70C for 5 minutes to inactivate T4 DNA ligase
5. Take 5L of connecting products and heat shock convert 50pL of competent cells, or take 1-2L of connecting products and shock convert 50pL of competent cells.
Note: If electroconversion is required, it is recommended to use ethanol precipitation method to remove T4 DNA ligase before electroconversion
iii Self cyclization of linear DNA
1. Reaction system: | Component | 50 μL Reaction system | Final concentration | | Linear vector DNA | X μL | 5-50 ng | | 10xLigation Buffer | 5 μL | / | | T4 DNA Ligase，5 U/uL | 1 μL | 5 U | | ddH20 | Add to 50 μL | 50 μL |
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2. Vortex oscillation, instant centrifugation, collects the solution on the pipe wall to the bottom of the pipe.
3. Reaction conditions: Incubate at sticky end 22C for 10 minutes, and incubate at flat end 22C for 1 hour.
4. Instantly centrifuge and collect the solution on the tube wall to the bottom of the tube. Incubate at 65C for 10 minutes or 70C for 5 minutes to inactivate T4 DNA ligase
5. Take 5pL of connecting products and heat shock them to convert 5pL of competent cells, or take 1-2pL of connecting products and shock them to convert 50pL of competent cells.
Note: If electroconversion is required, it is recommended to use ethanol precipitation method to remove T4 DNA ligase before electroconversion | 
