This is the protocol that the Dog Genome Project uses for RH mapping. The PCR recipe below is intended to work on the 118-cell line RHDF5000-2 panel (Vignaux et. al. 1999, Mammalian Genome) that we currently use. We run this panel in 1 1/2 plates (96 well) per marker with positive and negative controls. The column below titled "1 marker" shows the volumes used to run 1 marker on the plate and a half format. This recipe is optimized for the polymerase and thermocyclers that we use in the lab; you may have to optimize the reactions for your own conditions. For more information about the RHDF5000-2 panel, go to the University of Rennes, France Canine Radiation Hybrid Mapping Project Website.
PCR Mix Recipe:
| PCR Reagent: | 1 marker | Final Concentration |
| F-oligo at 20uM | 40 uL | 0.3 uM |
| R-oligo at 20uM | 40 uL | 0.3 uM |
| 10x PCR buffer | 240 uL | 1x |
| 1 mM dNTPs | 240 uL | 0.1 mM |
| 50mM MgCl2 | 72 uL | 1.5 mM |
| Taq (Bioline USA, 5U/uL) | 4.8 uL | 0.15U/rxn (0.01U/uL) |
| H2O | 163.2 uL | |
| Template DNA (10ng/rxn) | 10 uL/ well | 1ng/uL |
Dispense 5 uL per well for 144 reactions (1 ½ plates).
PCR program (using ABI GeneAmp 9600’s)
| 94°C | 5 minutes | ||
| 94°C | 20 seconds |  |
|
| 58°C | 20 seconds | 35 cycles | |
| 74°C | 20 seconds | ||
| 74°C | 2 minutes | ||
| 4°C | Hold |
If PCR gives too much background, increase Tm to 60°C; if signal is too weak, decrease Tm to 56°C.
Touchdown PCR protocol (if marker Tm indicates “TD” or a number range, e.g. 63-53)
| 94°C | 7.5 minutes | ||
| 94°C | 30 seconds | |
|
| 62°C (-0.5°C/ cycle) | 30 seconds | 20 cycles | |
| 72°C | 30 seconds | ||
| 94°C | 30 seconds | |
|
| 52°C | 30 seconds | 15 cycles | |
| 72°C | 30 seconds | ||
| 72°C | 2 minutes | ||
| 4°C | Hold |
If initial pcr gives too much background, increase touchdown to starting Tm
of 64°C and touchdown to 54°C