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:
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
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