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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
>chr1:63664766-63664926 161bp CCTGACCTCCGTTTTTGTGA TACCTACCCCTCGGAAGCAG
Mut= 157 bp
Wt= 161 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACTCGGTTTTTTTCTTAAAGCCATTGAAGGCAGTGTCATTGTGACCTCCATCGGGTGGGGGTGGGGTGGGAAAGAAAGATTAAAATAACCTGCCTGGGATTCTAAGAAGTCAGACAATAGACCTCTTCCCTTTGCTACCTCAATGCCTTCCTCCTGTGACTCTCCCGCCCCCCTTTCCTTTTTCATAGGTCCAACTCAAAGGTTCCATTTCACAAAATTTAAACTTACTTTTTTCTTTTGATTGTGCTGGTTCTTCTGTTCTTGGATGCCCAATAATTGTTGCTTGCCTGACCTCCGTTTTTGTGAGCATCATTACTCAGGGCACACTAAGTCCATTTTTCAGGGCTaacacgaggttgcctttgcagatcccgactgtcgtgggagagagctacacgttgcgtcccgtggaaagtgctatccacagctgcttccgaggggtaggtagcctggacgtccttaggagctggagcagacgtttgaagactttgcttaaattacgcatcacgctgattccaggacgacctgagtttagaggcagccttgccactagctttcagatgtctttagaaaacagacacggaaacagagggaagaaagggggcagattccccctgaatgtgtgtttcagcctcctcttgcttcccaggtgttgagctctggaatcaaggaagagaaatttctgtcttgggcacagtctgagcctctggtcctcttgtggctcccaacttgctaccgcctgtcagccgcggaaacagtcactcaccctgtgcggtgcagtgtctgcagaaccttcccgatcataggactgaggtaccatcaatatcatatttcccaaatggtcacttggcaaatgggcccggtggtggagttgcatagccccagactctctctgggatcctttctatacatctggaatgttcttgcctttagaaaattaattccctgtctgaggcggcatggttgaaaataaatgtatttccgataattagcgaaatattaagttttatctctggttcacactcggcttttcttctatagcagtggttcttcacttctatagcagtggtcgagatccctttggggtcgaatatcagatatcctgcatataagatatttacattatgattcataacagcaaaattacagttataaagtagcaacaaaaatacaTTTATCTTTGGGGGGCGGGGAGGCGTCACAACAGCGGGGAGCTGTATTAAAGGGTTGCAGCGTATTAAGGTTGAGAACCAAGGTATAAAACAGGTGTCTTCTGCTCCAAATAAACCTTTTAAACACTTCGGAGCTACCCATAAGGATGGTTTGTTAAGTGTGAAAGAAACATTCGGCTAAGCAGTTCACAGGGATCACGTTTCTTAAAATGTGGTACAAGCTGTACAAAAGGCTACGCTATTATCTCCAACTTCCTGAAACATTAAATTCCGTGGTTAGCAGGTAGTAGAGGGGCTGGTGAGATGGCTCAGTGGC
This mutation is a 826 bp deletion beginning at Chromosome 1 position 63,664,040 bp and ending after 63,664,865 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52654 | CCT GAC CTC CGT TTT TGT GA | Common | A | |||
| 52655 | TAC CTA CCC CTC GGA AGC AG | Wild type Reverse | A | |||
| 52656 | CAC CTG TTT TAT ACC TTG GTT CTC | Mutant Reverse | A | |||
| 52657 | Fluorophore-1 | AGA GAG CTA CAC GTT GCG TCC C | Quencher-1 | WT Probe | ||
| 52658 | Fluorophore-2 | AGG CGT CAC AAC AGC GGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52654 | 0.40 uM |
| 52655 | 0.40 uM |
| 52656 | 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.