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Stock No: 006678
Protocol 23720: Standard PCR Assay - Or8a1b<tm6Mom>
Version 3.2
Notes
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
Mutant = 400 bp
Heterozygote (Stock#006678) 475 bp and 400 bp
Heterozygote (Stock# 007766) ~450 bp and 400 bp
Wild type (Stock# 006678) 475 bp
Wild type (Stock# 007766) ~450 bp
Heterozygote (Stock#006678) 475 bp and 400 bp
Heterozygote (Stock# 007766) ~450 bp and 400 bp
Wild type (Stock# 006678) 475 bp
Wild type (Stock# 007766) ~450 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| mom0135 | GCA GCT GTG GGC TTG TTC TAT GGA TCC | Wild type | A | |||
| mom0136 | CAG TAA TAA GAC CAC AAA TGC CT | Common | A | |||
| mom0139 | GAG AAG CGC GAT CAC ATG GTC CT | Mutant | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| mom0135 | 0.50 uM |
| mom0136 | 0.50 uM |
| mom0139 | 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.