Reaction Mix (25ul):
| Tail DNA | 2.0ul |
| NTPs | 0.5ul |
| 10x PCR buffer | 2.5ul |
| miR F4 | 0.1ul |
| miR R5 | 0.05ul |
| AV 145 | 0.05ul |
| milliQ | 19.6ul |
| Taq (1/10) | 0.2ul |
10X PCR Buffer:
| 500 mM KCl |
| 100mM Tris pH 8.3 |
| 15 mM MgCl2 |
| 1 mg/ml BSA |
| 2 mM dNTPs |
| dH20 |
(store at -20º C)
PCR Primers: all used at 100um
| miR F4 | 5′ TCG AGT ATC TGA CAA TGT GG 3′ |
| miR R5 | 5′ TAG CCA GAA GTT CCA AAT TGG 3’ |
| AV 145 | 5’ ATA GCC TGA AAC CAA CTG TGC 3’ |
PCR Conditions:
| 1. | 94C | 5:00 |
| 2. | 94C | 0:30 |
| 3. | 55C | 1:00 |
| 4. | 72C | 1:30 |
| 5. | Goto 2 x34 cycles | |
| 6. | 72C | 5:00 |
| 7. | 4C | 0:00 |
| END | ||
Expected Bands:
| WT 250bp |
| MUT FL 289BP |
| Mut KO 441 bp |