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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
>chr4:99961303+99961413 111bp CTCAGTAACATGGTAGAGATATCCAAG GAAGAAATGCCAGGGGAAGA
Mut= 129 bp
Wt= 111 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGTCAGTCCACATTGAGAACCTTCCCCAGATGGACTGTCACACATGCACTGTGTTCTTGCCTTACAGCACCTCAGTGTATGGTTCCAAAGCTACAAAGTTCCATAGGGAAGAAGGGAAAGGAGGGAGGGCGGGCTTTGCTCAACAGCTCTGTTCTGTCTCAGTAACATGGTAGAGATATCCAAGATGaagatgtttctttaagaaagcatctttgctggtccaacagtgtcttcattgtccatgttcttcttcccctggcatttcttcgtgggagctttgtatttatatgttctggatttcctatcctagattggtcgcctggttattgggcagaatggaatactctccacccctgccgtatcttgcatcatccgaaaaatcaaagctattggtgggatcattctgacagccagccataatccaggagggcccaatggagattttggaattaaattcaatatttctaatgggggtgagtattctgtcatcttaaggacaagtagatacagttggataagctagtgcccgaagccctgggctctgattcccatcttaagaaggggttttgcttccctttggagatggtaaagtacttcttagtctacagctgccttctttgtaatgaattgtagctatcatggtattctactttagaaaagtctaactggtattattagctcctagcactttgaaggttgacacaggagAATTGTGAATTCAAGGCCAATCCAAACTGTATAGTGAGAATTTGACTCAGAAACATCTGGAAAAAAAAAAAAAAAAAAAAGAGTCCTGGTGCTTTGTGACTCAGTACTGGGGTCGAGGGATTGCTGAGTGCTGGGTTGTGTTTTCTCATGAACCGCA
This mutation is a 521 bp deletion beginning at Chromosome 4 position 99,961,333 bp and ending after 99,961,853 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 40551 | CTC AGT AAC ATG GTA GAG ATA TCC AAG | Common | A | |||
| 40552 | GAA GAA ATG CCA GGG GAA GA | Wild type Reverse | A | |||
| 40553 | TCA CAA AGC ACC AGG ACT CT | Mutant Reverse | A | |||
| 40554 | Fluorophore-1 | AAC AGT GTC TTC ATT GTC CAT GTT | Quencher-1 | WT Probe | ||
| 40555 | Fluorophore-2 | ATG AAT TGT GAA TTC AAG GCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 40551 | 0.40 uM |
| 40552 | 0.40 uM |
| 40553 | 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.