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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
>chr18:36997754+36997904 151bp CATTTAGCCACTGGATGTCG CTTCTGGTCGATTTCCTAATCTC
Mut = 159 bp
Wt = 151 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
ACATTTAGCCACTGGATGTCGCTGCCGTCCATGAAATGTATTGCTTTTAAGAACAAAGATATAgttcgctgtttctcaggggataggaagtagctacgttcggagatctcggcgtttcggttaaaaaatgagattaggaaatcgaccagaagatattagaacttgtgtgcatctcagatggcacattcatggacttttaagacaagagaatgcttcagtggtgatctccaaatgtctgaggcatggagcttggcgcctgctactttggcttctgctccttgcaacatgggatgtagggagcggccagcttcactactctgtcccagaggaggccaaacacggcaccttcgtgggccgcatcgcgcaggacttggggctggagctggctgaactggtgccccgcctgttcagggtggtgtccaaggaccgcggggaccttctggaggtaaatcttcagaatggcattttgtttgtgaattctcggatcgaccgggaggagctgtgcgggcagaacgcggagtgtagcatccacctggaggtgatcgtggacaggccgctgcaggttttccacgtggaggtggaggtgagggacattaatgacaaccctccgatattctccgtagcggaacaaaagatcttggtagctgaatctagactgcttgattctcgatttccactagaaggcgcctctgatgcagatgttggagagaactctatgcttacttacaaactcagttcaaatgagttttttatccttgatattgtaaacaaaagaggcaaaggcaaatttccagtgcttgttcttcgaaaactgatagatcgagaagaaaatcctcagctaaagttgctactgacagcaaccgatggaggcaaacctgaatttacgggatcggtttctctgttgatccaggtgttggatgttaatgacaatgctcctgtgtttgacagatctgtttacgaagttaagatgtatgagaatcaagagaataaaaccttagtaatatggctgaatgctactgactcagatgaaggaataaacaaagaagtggagtattcatttagctctttagcctcatcgattataagacagaaatttctaataaacgaaaagactggagagataaaaataaacggcgctattgactttgaggagagtaacaattatgaaattcacgtggatgctacagataaaggatacccacctatggttgctcactgcaccgtactcgtggaaatcttggatgaaaatgataacgcaccggagatagtccttacttctttgtctttgccggtgaaagaagatgctcctttgggctctgtaatcgccctgatcagcgtgtcagacaaagactctggtgtcaatggacaggtgacctgctccttgactaaccatgtccccttcaagttggtgtccaccttcaagaattactattccctcgtgctggacagcgccctggaccgagagaccacagctgactataaggtggtggtgacagcccgcgacgggggctcgccctctctgtgggccacggccagcgtgtccgtggaggtggctgacgtgaacgacaatgctcctgcattcgcacatcccgaatacacggtgttcgtgaaggagaacaacccgcctggagtgcacatcttcacagtattggcggtggatgctgacgcacaggagaacgcgctggtgtcctactcgctggtggagcggagggtgggcgagcgcttgctgtcgagctatgtgtctgtgcacgcggagagcggcaaggtgttcgcgctgcagcctctggaccatgaggagctggagctgctgcagttccaggtgagcgcgcgggatgctggtgtgcctgccctgggcagcaatgtgactctgcaggtgtttgtgcaggatgagaatgacaatccgcccacgctgctcggacatcagtcaggtggacctgctggcgaatttagccagcttgtttcgcggtcagtgggtgcgggacacgtggtgtcaaaggtgcgcgcggtggatgcggactccggttacaacgcgtggctgtcttatgagctccacccaactgtgggcgctcgcagccctttccgtgtcggtctctacacgggtgagatcagcatgacccgagccctggatgaatctgatttaccgcgccagcgcctattggtgttggtgaaggaccacggcgaacctatgttgatcgccactgccaccgttttggtgtccctggtggagaacggccaggtgccaaaagcttcttcccaggggttgcccaactcctcccgcagggaggcgtcgctgatggatgtcaatgtatacctgatcattgccatttgcgcagtgtccagcctgttggtgctcacgctgctgctatacacagccctgcgctgctcagctgtgcccatgcaggctgggtgcgggcttgggaagcccactctggtgtgctcaagcgctgtggggacctggtcatactcacagcagagacagcagcgagtatgctcaggagagggtccgcccaaaaccgacctcatggcttttagccccagtcttacaccttgcccagtggccgaagtgggaatggaaagtcattctgttggaggagacgtgcccggaaaggtgggctattactttttactctcatcttgcttgatgaaatacattttctgtctcattttattttatgacaaatgtgtgtcccagatgtatttttttttgtctgagtaccactatgcacaaGTATTTTTAACAATGCGTTGGAATATAGTTTATTCCAGCAATTGGTTTTAAGATGTCCATCTA
This mutation is a 2665 bp deletion beginning at Chromosome 18 position 36,997,816 bp and ending after 37,000,480 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51580 | CAT TTA GCC ACT GGA TGT CG | Common | A | |||
| 51581 | CTT CTG GTC GAT TTC CTA ATC TC | Wild type Reverse | A | |||
| 51582 | ACA AGT TTT AGG CAG AGG TTT G | Mutant Reverse | A | |||
| 51583 | Fluorophore-1 | CGC TGT TTC TCA GGG GAT AGG | Quencher-1 | WT Probe | ||
| 51584 | Fluorophore-2 | CGT TGG AAT ATA GTT TAT TCC AGC AAT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51580 | 0.40 uM |
| 51581 | 0.40 uM |
| 51582 | 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.