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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
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
>chr19:16873547-16873655 109bp GAAGAGCCTGGGAAGATGC TCTCAGCCGAATGCTGGA
Mut = 108 bp
Wt = 109 bp
Fam = Mut
Hex = Wt
Sequence
MUT Sequence (junction in uppercase):
acttaagcctggtatccggctttccgagaggcgttaaggcccactgtgggccggacggaagtggaagagcctgggaagatgccacgGTtaggggaccccgcgaccggtgagaaggcgcctgtcgagagactgagacaatggtcccagaccgagagggtgctccaggaaggtcagcaaactt
This mutation is a 1274 bp deletion beginning at Chromosome 19 position 16,872,358 bp and ending after 16,873,631 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50701 | GAA GAG CCT GGG AAG ATG C | Common | A | |||
| 50702 | TCT CAG CCG AAT GCT GGA | Wild type Reverse | A | |||
| 50703 | ACC TTC CTG GAG CAC CCT CT | Mutant Reverse | A | |||
| 50704 | Fluorophore-1 | TCC TAC AGC GAC CAA AAG CC | Quencher-1 | WT Probe | ||
| 50705 | Fluorophore-2 | CGG TTA GGG GAC CCC G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50701 | 0.40 uM |
| 50702 | 0.40 uM |
| 50703 | 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.