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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 = T
Wild type = CTC
>chr8:13878194+13878265 72bp AGCCTTTGACCCAGCAGAG AAGCTCGGGCTCGTTTTCT
Sequence
WT sequence (with mutation in parentheses):
GAAAGAGCAGCCGAGCAAAGACACCGAGTCTGGGAAAAGCCCTTCCCCTCCTGAGCGGCAGAACCCAGCCTTTGACCCAGCAGAGGCGAGGCCCACTCCAGCC(ctc/t)CCCATGGAAGCACAGAAAACGAGCCCGAGCTTGTGTCCAGAGTCACAGGCTTCTGGTCCTCCAGTTCTGGAGCCTCAGG
Mutation is del3 (ctc) and ins1 (t)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46254 | AGC CTT TGA CCC AGC AGA G | Forward | A | |||
| 46255 | AAG CTC GGG CTC GTT TTC T | Reverse | A | |||
| 46256 | Fluorophore-1 | CCA GCC CTC CCC ATG G | Quencher-1 | WT Probe | ||
| 46257 | Fluorophore-2 | CTC CAG CCT CCC ATG GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46254 | 0.40 uM |
| 46255 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.