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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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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
>chr9:40317649-40317754 106bp ACGCCCCTTTCTTCACTTG ATAGCGGATGCTGGGGTAG
Mutant= 108 bp
Wild Type = 106 bp
Sequence
Wt Sequence: acgcccctttcttcacttggcctcccagagACtatgggaagagagcttatctcgtcgtctttccaacacacaagcatccgccagcctctaccccagcatccgctat
Mutant Sequence: acgcccctttcttcacttggcctcccagagATggtagtagaaggttggaaatcaatgaaacagccactggcctttttaaaaatcctgccagtcgttggctgtcatact
474 bp deletion beginning at Chromosome 9 negative strand position 40,317,723 bp and ending after 40,317,250 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 36240 | ATA GCG GAT GCT GGG GTA G | Wild type Reverse | A | |||
| 36241 | ACG CCC CTT TCT TCA CTT G | Common | A | |||
| 36243 | AGT ATG ACA GCC AAC GAC TGG | Mutant Reverse | A | |||
| 36244 | Fluorophore-1 | TGA AAC AGC CAC TGG CCT | Quencher-1 | MUT Probe | ||
| 36245 | Fluorophore-2 | CGT CGT CTT TCC AAC ACA CA | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 36240 | 0.40 uM |
| 36241 | 0.40 uM |
| 36243 | 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.