Recombination
ClonExpress II One Step Cloning Kit C112(Vazymebiotechco.,ltd)was used for recombination.
1. Calculation of the amount of vectors：
The optimal amount of vector for the recombination with ClonExpress II is 0.03 pmol, while the optimal amount of insert is 0.06 pmol (molar ratio of vector to insertion is 1:2), as roughly calculated as follows:
The optimal mass of vector = [0.02 x number of base pairs] ng (0.03 pmol)
The optimal mass of insert = [0.04 x number of base pairs] ng (0.06 pmol)
For example, when cloning an insert of 2 kb to a vector of 5 kb, the optimal mass of vector is 0.02 x 5000 = 100 ng, and the optimal mass of insert is 0.04 x 2000 = 80 ng.
a.When using digested vectors and amplified inserts directly for recombination (without purification), the total volume of vectors and inserts should be ≤ 4 μL (1/5 of the total volume of recombination reaction system).
2. Calculate the amount of DNA for recombination by formula. Dilute linearized vectors and inserts before recombination to make sure the loading accuracy. The volume of each component loaded should be no less than 1 μL.
3. Set up the following reaction on ice:
a.X or Y indicates the amount of vector or insert calculated by formula
4. Gently pipette up and down for several times to mix thoroughly (DO NOT VOTEX!). Spin briefly to bring the sample to the bottom of the tube before reaction.
5. Incubate at 37 °C for 30 min and immediately place the tube at 4 °C or on ice.
▲It is recommended to use an instrument with high accurate temperature controlling system (i.e. a PCR instrument) for the reaction. The recombination efficiency can reach its peak at 30 min. Longer or shorter reaction time will decrease on the cloning efficiency.
▲The recombination product can be stored at -20℃ for one week. Thaw the product before transformation.
1. Calculation of the amount of vectors：
The optimal amount of vector for the recombination with ClonExpress II is 0.03 pmol, while the optimal amount of insert is 0.06 pmol (molar ratio of vector to insertion is 1:2), as roughly calculated as follows:
The optimal mass of vector = [0.02 x number of base pairs] ng (0.03 pmol)
The optimal mass of insert = [0.04 x number of base pairs] ng (0.06 pmol)
For example, when cloning an insert of 2 kb to a vector of 5 kb, the optimal mass of vector is 0.02 x 5000 = 100 ng, and the optimal mass of insert is 0.04 x 2000 = 80 ng.
a.When using digested vectors and amplified inserts directly for recombination (without purification), the total volume of vectors and inserts should be ≤ 4 μL (1/5 of the total volume of recombination reaction system).
2. Calculate the amount of DNA for recombination by formula. Dilute linearized vectors and inserts before recombination to make sure the loading accuracy. The volume of each component loaded should be no less than 1 μL.
3. Set up the following reaction on ice:
| Components | Recombination |
|---|---|
| Linearized Vectors | X μL |
| Inserts | Y μL |
| 5 x CE II Buffer | 4 μL |
| Exnase II | 2 μL |
| ddH2O | to 20 μL |
a.X or Y indicates the amount of vector or insert calculated by formula
4. Gently pipette up and down for several times to mix thoroughly (DO NOT VOTEX!). Spin briefly to bring the sample to the bottom of the tube before reaction.
5. Incubate at 37 °C for 30 min and immediately place the tube at 4 °C or on ice.
▲It is recommended to use an instrument with high accurate temperature controlling system (i.e. a PCR instrument) for the reaction. The recombination efficiency can reach its peak at 30 min. Longer or shorter reaction time will decrease on the cloning efficiency.
▲The recombination product can be stored at -20℃ for one week. Thaw the product before transformation.
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