Reaction Mix:
| Tail DNA | 2.0ul |
| 10x buffer | 2.0ul |
| NII (20uM) | 0.2ul |
| NIII (20uM) | 0.2ul |
| 3’5′ (20uM) | 0.4ul |
| milliQ | 14.2ul |
| Taq (1/25) | 1.0ul |
PCR Primers:
| NII | 5′ GGT ATT GAA TTG AAG CAC 3′ |
| NIII | 5’ ATT CGC CAA TGA CAA GAC 3’ |
| 3’5′ | 5’ TTC AAT ACC TGC CCA AGG 3’ |
PCR Conditions:
| 1. | 94C | 3:00 |
| 2. | 94C | 1:00 |
| 3. | 52C | 1:00 |
| 4. | 72C | 2:00 |
| 5. | Goto 2 x33 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:
| Wt= ~230 bp |
| Mut= ~350 bp |