Reaction Mix:
| Tail DNA | 4.0ul |
| 10x PCR buffer | 2.0ul |
| k-5′-IO (20uM) | 0.2ul |
| K-3′-1a (20uM) | 0.1ul |
| K-3′-neo (20uM) | 0.25ul |
| milliQ | 12.45ul |
| Taq (1/25) | 1.0ul |
PCR Primers:
| k-5′-IO (5’WT) | 5′ AGG GTA GGT GTT GGG ATA GC 3′ |
| K-3′-1a (3’WT) | 5’ CTC AGT CAT TTT CAG CAG GC 3’ |
| K-3′-neo (3’MUT) | 5’ ACG AGA CTA GTG AGA CTG GC 3’ |
PCR Conditions:
| 1. | 94C | 3:00 |
| 2. | 94C | 1:00 |
| 3. | 60C | 1:00 |
| 4. | 72C | 1:00 |
| 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= ~360 bp |
| Mut= ~270 bp |