Summary
- Gibson Assembly
- Check with PCR
Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5.
There is no addition of vector this time.
|
1
|
2
|
Gibson Rxn mix
|
15 μL
|
15 μL
|
DNA fragments
|
5 μL
|
5 μL
|
DNA fragments
|
1
|
2
|
#1
|
1.0 μL
|
|
#2
|
1.0 μL
|
|
#3
|
1.0 μL
|
|
#4
|
|
1.0 μL
|
#5
|
|
1.0 μL
|
dH2O
|
2.0 μL
|
3.0 μL
|
Total
|
5.0 μL
|
5.0 μL
|
PCR Check
- Product 1: outer-most primers for whole assembly
- Product 1: Gibson primer pair for hPCD
- Product 2: outer-most primers
- Product 2: Gibson primer pair for hPCD
Reagent |
1 |
2 |
3 |
4
|
Gibson product |
0.5 μL |
0.5 |
0.5 |
0.5
|
primer 1 |
1.0 |
1.0 |
1.0 |
1.0
|
primer 2 |
1.0 |
1.0 |
1.0 |
1.0
|
2x GoTaq PCR mix |
14.5 |
14.5 |
14.5 |
14.5
|
dH2O |
12.0 |
12.0 |
12.0 |
12.0
|
Program:
- 95°C, 3 min.
- [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
- 72°C, 3 min.
- 4°C ∞
|