Table 2
Protocol of DOP–PCR
| Reagent | Volume (μl) | Reaction |
|---|---|---|
| 10× high salt buffer: 200 mM Tris/HCl (pH 9.2), 600 mM KCl, and 20 mM MgCl2. | ||
| 10× low salt buffer: 100 mM Tris/HCl (pH 8.4), 100 mM KCl, and 15 mM MgCl2. | ||
| DOP–PCR, degenerate oligonucleotide primed polymerase chain reaction. | ||
| Preamplification step | ||
| 10× high salt buffer | 0.5 | 1 minute denaturation at 94°C |
| 2 mM dNTP | 0.5 | 1 minute annealing at 25°C |
| 10 μM UN1 primer | 0.5 | 3 minute ramp from 25°C to 74°C |
| ThermoSequenase (4 U/μl) | 0.5 | 2 minutes extension at 74°C |
| Sample | 3.0 | 4 cycles in total |
| 5.0 (total) | ||
| Second amplification step add | ||
| 10× low salt buffer | 2.0 | 1 minute denaturation at 94°C |
| 2 mM dNTP | 5.0 | 1 minute annealing at 56°C |
| Water | 12.2 | 2 minutes extension at 72°C |
| 100 μM UN1-primer | 0.3 | 30 cycles in total |
| Taq polymerase LD (5 U/μl) | 0.5 | |
| 25.0 (total) | ||
