1.1 1st PCR
Followings are the 1st PCR solution and PCR program
Below PCR program is based upon using PCR enzyme included with our BCF4050 product. If you are using PSS4050, follow the manual of PCR enzymes which can perform 2 step PCR cycle such as Toyobo’s KOD-Plus-NEO, KOD Hot Start DNA Polymerase, and/or KOD Hot Start Master Mix
1st PCR Solution
| Reagents | |
| 10x PCR buffer | 5 μℓ |
| 2 mM dNTPs | 5 μℓ |
| 25 mM MgSO4 | 3 μℓ |
| 10 μM 1st-ORF-F | 1 μℓ |
| 10 μM 1st-ORF-R | 1 μℓ |
| Plasmid DNA | 1 ng |
| PCR NDA Polymerase | 1 μℓ |
| Ultra pure water | 33 μℓ |
| Total | 50 μℓ |
1st PCR Program
| Seg. | Temp. | Time | Cycle |
| 1 | 94 °C | 2 min | 1 |
| 2-1 | 98 °C | 10 sec | 30 |
| 2-2 | 68 °C | 30 sec/kb* | |
| 3 | 20 °C | – | – |
* for 1 kb or less, set 1 min.