1.2 2nd PCR
Followings are the 2nd PCR solution and PCR program.
Please note that the composition differs for N-terminal tags and C-terminal tags. Also some primers have different concentrations. (in red)
The PCR program is common for N-terminal tags and C-terminal tags.
Following PCR program and solutions are based on PCR enzyme included with our BCF4050. If you are using PSS4050, follow the instructions for a PCR enzyme (e.g KOD-Plus-NEO, KOD Hot start master mix, KOD Hot start DNA Polymerase, etc.) that can perform a two-step PCR cycle.
2nd PCR Solution for N-term tagged
| Reagents | |
| 10x PCR buffer | 5 μℓ |
| 2 mM dNTPs | 5 μℓ |
| 25 mM MgSO4 | 3 μℓ |
| 10 μM 2nd_NF2 | 1 μℓ |
| 100 nM 2nd_NF1 | 1 μℓ |
| 100 nM 2nd_NR1 | 1 μℓ |
| 10 μM 2nd_NCR2 | 1 μℓ |
| 1st PCR product | 1 μℓ |
| PCR NDA Polymerase | 1 μℓ |
| Ultra pure water | 31 μℓ |
| Total | 50 μℓ |
2nd PCR Solution for C-term tagged
| Reagents | |
| 10x PCR buffer | 5 μℓ |
| 2 mM dNTPs | 5 μℓ |
| 25 mM MgSO4 | 3 μℓ |
| 10 μM 2nd_CF1 | 1 μℓ |
| 100 nM 2nd_CR1 | 1 μℓ |
| 10 μM 2nd_NCR2 | 1 μℓ |
| 1st PCR product | 1 μℓ |
| PCR NDA Polymerase | 1 μℓ |
| Ultra pure water | 32 μℓ |
| Total | 50 μℓ |
2nd PCR Program ( for both N-term tagged and C-term tagged)
| Seg. | Temp. | Time | Cycle |
| 1 | 94 °C | 2 min | 1 |
| 2-1 | 98 °C | 10 sec | |
| 2-2 | 60 °C | 1min | 5 |
| 2-3 | 68 °C | 30 sec/kb * | |
| 3-1 | 98 °C | 10 sec | 30 |
| 3-2 | 68 °C | 30 sec/kb * | |
| 4 | 72 °C | 2 min | 1 |
| 5 | 20 °C | – | – |
* When 1 kb or less, set to 1 min.