Team:NCKU Tainan/Protocol
Protocol
1. Gently mix the following reaction by pipetting and centrifuge briefly.
| 20 μl system | 50 μl system | |
|---|---|---|
| Template | 12～20 ng | 30～50 ng |
| Forward primer | 1.0 μl | 2.5 μl |
| Reverse primer | 1.0 μl | 2.5 μl |
| dNTP | 1.6 μl | 4.0 μl |
| 10x Buffer | 2.0 μl | 5.0 μl |
Program of KOD DNA polymerase
| Temperature | Time | |
|---|---|---|
| 94 ℃ | 3 min. | 25～30 cycles |
| 94 ℃(Denaturation) | 40 sec | |
| 57.5 ℃(Annealing) | 30 sec | |
| 72 ℃(Extension) | Depend on sequence size(2 kbp/min. for Taq) | |
| 72 ℃ | 5 min. | |
| 4 ℃ | ∞ |
2. Confirm the size of the digested product by gel electrophoresis. 3. Gel purification of the target size.
1. Digestion (vector)
| Plasmid | 200 ng | 1000 ng |
|---|---|---|
| EcoRI / SpeI | 0.2 μl | 1 μl |
| XbaI / PstI | 0.2 μl | 1 μl |
| CutSmart Buffer | 2 μl | 5 μl |
| ddH2O | Up to 20 μl | Up to 50 μl |
| Digestion at 37℃ for 2.5hr. |
2. Digestion (insert)
| Plasmid | 200 ng | 1000 ng |
|---|---|---|
| EcoRI / XbaI | 0.2 μl | 1 μl |
| SpeI / PstI | 0.2 μl | 1 μl |
| CutSmart Buffer | 2 μl | 5 μl |
| ddH2O | Up to 20 μl | Up to 50 μl |
| Digestion at 37℃ for 2.5hr. |
3.Confirm the size of the digested product by gel electrophoresis. 4. Gel purification of the target size. 5. Ligation
| Vector (2 kbp) | molar ratio = 1:3(can up to 1:10 depending on the DNA sizes) |
| Insert (1.5 kbp) | |
| Quick Ligase Reaction Buffer (2X)* | 10 μl |
| Quick Ligase | 1 μl |
| ddH2O | Up to 20 μl |