Table 1.
Touchdown PACAP PCR genotyping protocol.
| PCR primers (Invitrogen Cat. # 10336022) | ||||
| omPCPF1+: GTT ATG TCG GTG CGG AGG AGT TTC | ||||
| omPCPD2−: GCT GGA TAG TAA AGG GCG TAA | ||||
| omPCPF2−: TTC AAG CAG CCT GCC CCA GAC TCA | ||||
| RXN | 1X | |||
| DNA | 150 ng/sample | |||
| MgSO4 | (25 mM) | 6 µL | ||
| 5X Buffer, pH 9.15 |  |
10 µL | ||
| 6 mM MgSO4 | ||||
| dNTP’s mix | (2.5 mM each) (Ambion cat #: 8228 G) | 4 µL | ||
| *omPCPF1+ (10 µM) | 3 µL | |||
| *omPCPF2− (10 µM) | 3 µL | |||
| *omPCPD2− (10 µM) | 1.5 µL | |||
| DMSO | 1.0 µL | |||
| Titanium Taq (BDBiosciences Clontech cat #: 639208) | 0.4 µL | |||
| Pfu Native (Stratagene cat #: 600136) | 0.1 µL | |||
| ddH2O | 19 µL | |||
| TOTAL | 50 µL | |||
| PCR program: | ||||
| 95 °C - 5 mins | ||||
| 94 °C – 1:00 min |  |
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| 70 °C – 30s | ||||
| Decrease temp by 1°C every 1 cycle | ||||
| 72 °C – 4:30 mins | ||||
| 94 °C – 1:00 min |  |
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| 60 °C – 30s | ||||
| 72 °C – 4:30 mins | ||||
| 72 °C – 7 mins | ||||
| 4 °C – 20 mins | ||||
| 25 °C – ∞ | ||||
| EXPECTED FRAGMENT SIZES:WT 1.3 Kb; mutant 1.7 Kb | ||||