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Notes
Melting curve analysis is done using a Roche Light Cycler 480.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
HET 88C +/-1.0 area 3
HET2 91C +/-1.0 area 5
HOM 88C +/-1.0 area 8
WT 91C +/- 1.0 area 8
HET2 91C +/-1.0 area 5
HOM 88C +/-1.0 area 8
WT 91C +/- 1.0 area 8
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0837 | CCG GTG GAT GTG GAA TGT GTG | Mutant | A | PGK R | ||
| oIMR0838 | AGA GCT CCA GGC ACA AGG GC | Common | A | |||
| oIMR0839 | AAC GGG CCA GAC CTC GGG T | Wild type | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR0837 | 0.50 uM |
| oIMR0838 | 0.50 uM |
| oIMR0839 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.