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 |
6. Transform the product by heat shock.