Radiation Hybrid Mapping PCR Protocols

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:

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

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