Protocols for amplification of microsatellite markers:
Markers FH2010, FH2054, Pez05, Pez06, Pez08, Pez10, Pez11 and Pez12 were run at 58o annealing temperature and 1.5mmol MgCl2 concentration. Markers FH2079, Pez01, Pez13, Pez15, Pez16, Pez17, Pez20, and Pez21 were amplified using a 55o annealing temperature and 2.0mmol MgCl2.
| single reaction: 1.5 mmol MgCl2 | single reaction: 2.0 mmol MgCl2 | |
| AmpliTaq Gold 10x Buffer | 1uL | 1uL |
| dNTP mix (1mmol) | 1uL | 1uL |
| MgCl2 solution (50mmol) | 0.3uL | 0.4uL |
| F/R primer mix (10umol each) | 0.1uL | 0.1uL |
| diH2O | 2.45uL | 2.35uL |
| AmpliTaq Gold (5U/uL) | 0.05uL | 0.05uL |
| 6-FAM labeled primer (10umol) | 0.1uL | 0.1uL |
| Genomic DNA (2.5 ng/uL) | 5uL | 5uL |
| Total reaction volume | 10uL | 10uL |
| PCR program (ABI 9700): | ||
| 94o 10 min | ||
| 94o 30 sec 58o or 55o 30 sec 72o 30 sec |  | 35 cycles |
| 72o 10 min | ||
| 4o ∞ | ||
3uL of the amplified PCR product were added to 10 uL of ABI HiDi Formamide with 0.1uL of GeneScan 500 Liz Size Standard (ABI). Samples were denatured at 95oC for 5 minutes then transferred directly to ice and cooled for 5 minutes. Denatured samples were put directly onto the ABI 3730 for electrophoresis.
Protocol for sequencing mtDNA:
| PCR: | single reaction |
| AmpliTaq Gold 10x Buffer | 1uL |
| dNTP mix (1mmol) | 1uL |
| MgCl solution (50mmol) | 0.3uL |
| F/R primer mix (10umol each) | 0.1uL |
| diH2O | 2.55uL |
| AmpliTaq Gold (5U/uL) | 0.05uL |
| Genomic DNA (2.5 ng/uL) | 5uL |
| Genomic DNA (2.5 ng/uL) | 5uL |
| Total reaction volume | 10uL |
| PCR program (ABI 9700): | ||
| 94o 10 min | ||
| 94o 30 sec 58o 30 sec 72o 30 sec | | 35 cycles |
| 72o 10 min | ||
| 4o ∞ | ||
| Clean-up: | single reaction: |
| Exo | 1uL |
| SAP | 0.5uL |
| Dilution buffer | 0.5uL |
| PCR Rxn (from above) | 4uL |
| Total reaction volume: | 6uL |
| Clean-up protocol (ABI 9700): |
| 37o 30 min |
| 80o 15 min |
| 4o ∞ |
| Sequencing: | single reaction: |
| ABI BigDye Terminator rxn mix | 1uL |
| ABI Sequencing Buffer | 0.6uL |
| Primer - 1.5uMol | 1.4uL |
| Clean PCR Rxn (from above) | 2uL |
| Total reaction volume: | 5uL |
| Sequencing protocol (ABI 9700): | ||
| 96o 1 min | ||
|
96o 10 sec 50o 5 sec 60o 4 min |
|
25 cycles |
| 4o ∞ | ||
Add 10 uL of water to each reaction, filter through pre-moistened Sephadex columns, and transfer to the ABI 3730 for elcrophoresis.
