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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
>chr7:46997246-46997347 102bp AGTGGAAAAGAGGCTTATCTAGGAC ACCAGCTGCGTCCAGTCA
Mut= 104 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GAGACTTAGCTCTGTTTGTCATGCTTCTTTAATAGGTATTTAAGAGATTGAAGTCTTTCTTTCAGGGTTCAAATCATCCTTATTCGTGTTTCAGTCACTTTCTTTACGCAGTTACTTAAAAACCAAAGTCCACACAAATAGCATCTGTGCCCGGGCAGGATTCATGACCTTGGGATCTCGGAGAGCAGtgtaaaggagcttgtgtagcaaattaatagttaatgaacttatatgccgcagttgaagagtccagagagctgtgctctttccttagattccttcccttcctcttccttcctttcctcgctttgttttctgtgttttctgcagtactggctggggattgaactcctagctgataaatgtcagacaagcactcttcctctgagcctcacatcccagcccctagactatatatttgtaaagtgttgctgcttatagcttttattctgcttttgtttttagaaaaggtacagtttggcggtggggcctccaaagaaagacccaaaagtgaaaggtgtccagtcggcagctgtgcaagctttccttaggcggaaagaagaggagcttagacaaaaaggtatgcgcttgtactgcatctcagcctgtctgccaccaaatccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaaacatcctgacagcttagtagtttggggatgttgtatgtgtgtctgggttaactttatagacaaatatgtgcgtgcatgtttgtaaatgtgctcataggttggttatttgggttgttttttctaaccccatctcacctacccccaaggctttgtggagctggccacagtgatattcacctgtcatcccaggtattcaacaggctgagacaagatcatttattgagctcaggagcttcagagtagctgggacaacgcatcaaggccttatctcttgaaggaaggtggggagacatgaaaaggcctggtgtatcaaccatttaactagccataatatgtctccaattctttaccttggtcctggcattgttatagatattcattgattcactcttaacagctccagtcagtgctggtgtcctattgacatggtatatttatgagccattttgtgcattttaagcaaaagccaagtcttttatctgcttagtcataaagagaagctcatatctgacccacttgtgtagtaattactcagttttgttagatgacttcctcatttgacacagctctctagaaaccataactcagattgagctttgactgtggagtggcagcacatgagcgctgctatgctgacactgttgaggtgatgtgctcttgttttattcctttcagccttggaagagaaaaaaaggaaagaggaactagtgaaaaagcgaattgagctcaaacatgacaaaaaagcaagagccatggccaagaggacaaaggacaattttcatggttacgatgggattcctgttgaggaaaagacaaagaagaagcagctggtggagagccacctgaaccagggaactgaccaggagtatgatgtggaggaggaagacttcatagactacaatcaggctgagctggaccaggactatgaggaggagcaagagccacccaaggctgaaagcaaaccaaaggccccccttaaaagtgccccgtcacccatgaacttcactgacttactgaggctagctgagaagaagcagtttgaaccagtggagatcaaggtagtgaagaaagcagaagaccggcccctgactgcagaggaactgagggagcgagagtttcttgaacgaaagcataggaaaaagaaacctgaacccgatgccaaactacctccacccgtgttgaaaaaggcgccctctcataaagacatcatgggtacaaagcccagcagaggtgctggggacaggcagcttgcttccaaaggattgccctttcctcaagctgagaagaaattcagacccagcacggccagtgagaaacaagcagctttgtcttcacccaaatccctgccaggcgagaggaccaaggtagggtctggcagtagcacccagccctcacttcgggagggtcacaacaggcctgtcttcaatggggctggaaagccccgccccagcacctgttcaccaagtgtcccaaagactcctgctagcgggactcagaaatctgcttctgagcacaaagccaaaaaacctcttccttctcatccgagccattccaagcctggacccacggttctctcacacaacaaatctaagagtccaggtgtcagacaaccaggcagcaactctggctctgctcctgggcagcctaacccagggacagctcgacccacacttagttctggccctgtgcccaggcgccagaacggcagctccagctcaggacctgagcagtcagccggtgggatcagaaagctggccagcaattcacatctctccggacgaacactcaacggcacaaatggtcctggacgacctgcaagcagctcaagtggccctgggcgacccatcagtggatctgctggttctgggagacctgtgggcagttctggaggccccgggcagcctgtaaacaatccacatgaccttcgacgaccaatgaacagtcttagctctcctgggcgggcagtcagtggccctgggagatccataagtggttccattccagctggacgaactgtcaattcaggccctggaagaccagtgagcagcctaggccctggacgagcagttagtaacccaggcctccccacaaagcccagatgtactgttgtctcagaaacaatttcttcaaagaatattattagtcggtcaagtaatggacagataaatggaatgaaacctctcctctctggctacagatctgctcaaggtaaagttgacccttatcccaaagagtatatttatcactatgcctttggagaaagtacctagctttatataatcaggaaccctatgaccactttcacaagccctgtaatgtgctcattgttgatagtggaaaagaggcttatctaggacccgctgttcacaccccccacctctttctcacatgcagtattgtgatgtgtgagAGGGGGTGACTGGACGCAGCTGGTGGTTTGTAGGATAATGTTCTACATGAAGACTAAGCCAGATGAGAGGATGATTAAGAACTTAGAGCCTTTGCTGCTCTGTCATTCAGCAGGTGTTTATTGAACACCTCCCCTGTAGCAGCTGCTATTTTAGACTGTCCTGAGTTCTATTCCTTTTCATGGTGGTG
This mutation is a 2893 bp deletion beginning at Chromosome 7 position 46,997,270 bp and ending after 47,000,162 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46823 | AGT GGA AAA GAG GCT TAT CTA GGA C | Wild type Forward | A | |||
| 46824 | ACC AGC TGC GTC CAG TCA | Common | A | |||
| 46825 | CAG TTA CTT AAA AAC CAA AGT CC | Mutant Forward | A | |||
| 46826 | Fluorophore-1 | CCC CAC CTC TTT CTC ACA TG | Quencher-1 | WT Probe | ||
| 46827 | Fluorophore-2 | AGG ATT CAT GAC CTT GGG ATC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46823 | 0.40 uM |
| 46824 | 0.40 uM |
| 46825 | 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.