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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 = 450 bp
Heterozygote = 450 bp and 237 bp
Wild type = 237 bp
Sequence
Seq with 10559 and oIMR9535
Mut sequence
WT sequence
GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG
GCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGATCCACTAGTTCTAGCCT
CGAGGCTAGAGCGGCCGTAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAA
GTATAGGAACTTCACCGGTACCTTACAAGCCGAATTCGCTAGCGGCGCGCCGG
ATCCACTCTGCACCAAGGGACAGCGCTGTCCAGTGTGGAGAACTTTACTGCTA
GGATTTCCAATTGTTAGGAACGTCGTTAGCGCGCGGGAGAGCGAAGGTAGGAC
TCCCGGCTTCTTTCTCCGGATGGGGGGGTTGGTTTCGTTCGCCCCTCCCGGTC
CTCGGAGACCCCGCGCCCCCCATTTTCGCCGCTTCGGATTCTTGGACCAGA
Mut sequence
WT sequence
GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG
GCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGATCCACTAGTTCTAGCCT
CGAGGCTAGAGCGGCCGTAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAA
GTATAGGAACTTCACCGGTACCTTACAAGCCGAATTCGCTAGCGGCGCGCCGG
ATCCACTCTGCACCAAGGGACAGCGCTGTCCAGTGTGGAGAACTTTACTGCTA
GGATTTCCAATTGTTAGGAACGTCGTTAGCGCGCGGGAGAGCGAAGGTAGGAC
TCCCGGCTTCTTTCTCCGGATGGGGGGGTTGGTTTCGTTCGCCCCTCCCGGTC
CTCGGAGACCCCGCGCCCCCCATTTTCGCCGCTTCGGATTCTTGGACCAGA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 10559 | TCT GGT CCA AGA ATC CGA AG | Common | A | |||
| 11122 | CTC CTC CGT GTC CTC GTC | Wild type Forward | A | |||
| oIMR9535 | GGG AGG ATT GGG AAG ACA AT | 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 |
| 10559 | 0.50 uM |
| 11122 | 0.50 uM |
| oIMR9535 | 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.