Reaction Mix:
| Tail DNA | 1.0 µL |
| 10x buffer (with NTPs) | 2.0ul |
| p53X6.5 (20uM) | 0.4 µL |
| p53X7 (20uM) | 0.4 µL |
| Neo18.5 (20uM) | 0.4 µL |
| dH20 | 14.8 µL |
| Taq (1/25) | 1.0ul |
PCR Primers:
| p53X6.5 | 5′ ACA gCg Tgg Tgg TAC CTT AT 3′ |
| p53X7 | 5′ TAT ACT CAg AgC Cgg CCT 3′ |
| Neo18.5 | 5′ CTA TCA ggC ATA gCg TTg g 3′ |
PCR Conditions:
| 1. | 94C | 3:00 |
| 2. | 94C | 1:00 |
| 3. | 60C | 2:00 |
| 4. | 72C | 2:00 |
| 5. | Goto 2 x30 cycles | |
| 6. | 72C | 5:00 |
| 7. | 4C | 0:00 |
| END | ||
10X PCR Buffer:
| 500 mM KCl |
| 100mM Tris pH 8.3 |
| 15 mM MgCl2 |
| 1 mg/ml BSA |
| 2 mM dNTPs |
| dH20 |
(store at -20C)
Expected Bands:
| Wild type (gene intact) = 430 bp on 1-2% gel |
| Mutant (gene knockout) = 590 bp |