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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 = 232 bp
Heterozygote = 232 bp and 357 bp
Wild type = 357 bp
>chr12:86817147+86817503 357bp GTCCGGTTCCTTCTATGCAG GAACCTTCGAGGGAAGACG
Sequence
MUT Sequence:
TACATCACGGGGGAGGCAGAGGGTTTCCCTGCCACAGCTTGAGGCGGCCGCGAAGTTCCTATACCTTTTGAAGAATAGGAACTTCGGAATAGGAACTTCGTCGACAAAGGATCCTTCTCGCGGCCGCGCAGTCAGAGAAGGCAAGGCAGCCGGCATCCAGACGTGCCACTGCCCGAGCTGGTGCATTACAGAGAGGAGAAACACGTCTTCCTTCTAAGGTTCA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37830 | GTC CGG TTC CTT CTA TGC AG | Wild type Forward | A | |||
| 37831 | GAA CCT TCG AGG GAA GAC G | Common | A | |||
| 37832 | CCT TGC AAA AGT ATT ACA TCA CG | Mutant Forward | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 37830 | 0.50 uM |
| 37831 | 0.50 uM |
| 37832 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.