| |
Set 1 |
Set 2 |
Set 3 |
Set 4 |
Set 5 |
| Reaction Mixture (reaction volume – 25µl ) |
|
Reaction Buffer (10X)a |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
|
MgCl2 (50mM)a |
2.0 |
2.0 |
2.0 |
2.0 |
1.5 |
|
dNTP Mix (each) (10mM)a |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
|
DNA Polymerase (1U)a |
1.5 |
1.0 |
1.0 |
1.0 |
1.0 |
|
Primers (each) (nM) |
Refer Table 3 |
|
First-PCR product (mL) |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
| Cycle Condition |
|
|
|
|
|
| Pre-denaturation (°C:min) |
94:4 |
94:4 |
94:4 |
94:4 |
94:4 |
|
Denaturation (°C:sec) |
94:30 |
94:30 |
94:30 |
94:30 |
94:30 |
|
Annealing (°C:sec) |
60b:30 |
58c:30 |
64:30 |
58c:30 |
58:30 |
|
Extension (°C:sec) |
72:30 |
72:30 |
72:30 |
72:30 |
72:30 |
| Number of cycles |
16 |
16 |
15 |
16 |
15 |
| Final extension (°C:min) |
72:7 |
72:7 |
72:7 |
72:7 |
72:7 |
| Hold (°C:min) |
20:∞ |
20:∞ |
20:∞ |
20:∞ |
20:∞ |
a Biotools Company (Spain)
b Touchdown from 60°C to 53.6°C (reduce by 0.4°C every cycle)
c Touchdown from 58°C to 52°C (reduce by 0.4°C every cycle) |
|
