| Product Description | 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. | |
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 |
|
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 |
|
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 |
|
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
|
|---|
