| | PCR amplification of cDNA segments by 2 stage nested PCR protocol |
| | Protocol: NESTED PCR |
| | Template: 50 ng cDNA |
| | Primer: each 0.5 uM |
| | dNTPs: each 0.2 mM |
| | PCR Buffer: 5 ul [10X, 100 mM Tris-HCl (pH 8.4), 500 mM KCl], |
| | Mg 2+ 1.5 mM, |
| | Sigma Taq Polymerase: 0.025 U/ul |
| | |
| | - STANDARD AMPLIFICATION - |
| | 1st round: |
| | size: 1192 bp |
| | Initial incubation: 94 degrees C for 4 min |
| | Denaturation: 94 degrees C for 45 s |
| | Annealing: 58 degrees C for 45 s |
| | Polymerization: 72 degrees C for 45 s |
| | PCR Cycles: 36 |
| | |
| | 2nd round: |
| | Template: 5 ul of 1st round product |
| | Primer: each 0.3 uM |
| | dNTPs: each 0.2 mM |
| | PCR Buffer: 5 ul [10X, 100 mM Tris-HCl (pH 8.4), 500 mM KCl], |
| | Mg2+ 1.5 mM, |
| | Sigma Taq Polymerase 0.025 U/ul |
| | |
| | -STANDARD AMPLIFICATION - |
| | Size: 271 bp |
| | Initial incubation: 95 degrees C for 2 min |
| | Denaturation: 95 degrees C for 45 s |
| | Annealing: 60 degrees C for 45 s |
| | Polymerization: 72 degrees C for 30 s |
| | PCR cycles: 36 |
| | Thermal Cycler: MJ Research PTC 100 |
| | 2 stage nested PCR reaction was performed. The product from the second |
| | stage was analysed by fluorescent DNA sequencing |
