Table 4.
PCR components and thermal conditions for the first insert.
| Item | Concentration | Amount | Temperature | Time | Cycle |
|---|---|---|---|---|---|
| pCBC_DT1T2 | (5 ng) | 94 °C | 2 min | 1 | |
| DT1-BsF1 * | 20 μM | 1 μL | 94 °C | 15 s | 30 |
| DT1-F1 * | 1 μM | 1 μL | 60 °C | 20 s | |
| DT2-R1 * | 1 μM | 1 μL | 68 °C | 20 s | |
| DT2-BbR1 * | 20 μM | 1 μL | 68 °C | 2.5 min | 1 |
| 10X KOD Buffer | 5 μL | 4 °C | Hold | 1 | |
| dNTP | 2 mM | 4 μL | |||
| MgSO4 | 25 mM | 3 μL | |||
| KOD-Plus- | 1 U/μL | 1 μL | |||
| Distilled Water | to 50 μL |
* Primers must be substituted for the second and third insert fragments. In this case, the concentrations of DTsc-BbR and DTsc-AaR are 21 μM.