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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
>chr17:34897088+34897222 135bp CTGGGGAAGTGGGAAAGTAGA CCCTCCGCTACTCACTGTTC
Mut = 105 bp
Wt = 135 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CCACCCCTTTATCCATTCTCCCGTGGGCGCGCGGGCTGCGGGACTGGGGGGTGTGGACGCCGCTGCCCCGCCCCCGGGAACCACGCCTCCCTCCTCGGAGGGACCGCGCCTCCTTggaattggggaaactgagggaggcgcgccaagactagggaggtagggatgaggggtctggagccttggcctccgacagccagagacctttgtctctgactcgtttccttcccacccagggcagtccactcccgccacaatggcctctggggtggaagtcctgcgcttccagctgcctggccacgaggccgcaacgttgcggaacatgaaccagctccgtgcagaggagcggttctgtgatgtgaccatcgtggccgacagcctcaaattccgtggccacaaggtcatcctggccgcctgctcaccgttcctccgggaccagttcttgctgaaccccagttctgagctgcaggtctcactgatgcacagcgcgcgtattgtggcggacttgctcctctcttgctacacaggcgccctggagttcgcagtcagggacatcgtgaactacctgacagcagcctcctacctgcaaatggagcatgtagtggagaaatgccggaacgcccttagccagttcattgagcccaaaataggccttaaagaagatggggtcagcgaggctagcctcctaagcagtgtcagtgccaccaagtccctcctccctccagccaggaccccaaagccagctccaaaacccccaccaccacctcctctaccccctccgctcctgcgaccggtgaagctggagtttccgctggatgaggacctagagctgaaggcggaagaggaggatgaggatgaagatgaagatgtttctgacatctgcatcgtcaaagtggagtcggctctagaagtggcacaccggctcaaaccccctggaagcctagccgggggtctgggtatcggggcctctgtgagcagccacctgggagagctggcccagagtagtgtggcccccaacactgttaccccaccgcaaggggtggtgaaggcatgctacagcctgtcagaggacgcggaaggggaaggcctgttgttgatcccaggaggccgggccagtgtgggggccacctcgggcctagtggaagcagcagcggtggccatggctgcccggggggcggggggcagtctgggggcagggggtagccggggacctctgcctgggggcttctcaagtggaaaccccttaaagaacatcaaatgcaccaaatgccctgaagtgttccagggtgtggagaagctggtcttccacatgcgtgcgcaacacttcatcttcatgtgcccacgctgcggcaagcagttcaaccacagcagcaacctcaaccgccacatgaacgtccaccgtggtgtcaagtcccattcatgtggcatctgcggcaagtgctttactcagaagtccacactgcacgatcacctcaacctgcactcaggagcccggccctaccggtgctcctactgtgacgtgcgctttgcccacaagcccgccattaggcggcacctcaaggagcagcatggcaaaaccacagctgagaacgtgctggaagctggtgtggctgagatcaatgtcctcatccgctgaaaggccaagaggctgggggcggtcctgggcccggcagaattggggaggggcggggtttgagggcaggcaaacattttgagggaggaagactggagagagagagagaccatgcctcaaagagcagaagaaaaagatggagccggagacagggttgataaggctgagggaatgtgggatcccaaagaaagatttcttttttgaagaggtgcataaacgatgtggaggaagagagggaaaaaaaaaaaaaggcaaagttgaaagttttggagaacagctctttcctggctaaagctggctgggggggggggggattctagcgtttcttgagatggggtgggggtgggaattgtagggggattgccaggaagcagcttacttgcccctggttagaatagatgacagggagaagggagcggagggcgacacatgtatgaagtaccagaaggaagggaggagatttttgcatgctggggaagtgggaaagtagatacaaggctcaccggaaatgaaatgatatgtgccaggtaagagaacaCGGTGGGCAACCTGGGTAGGTAATGAGACATTAGGCTACAGTAGCGAGAACAGTGAGTAGCGGAGGGGTGGGGGGGCAGGGCACTTAGAAAAAAAGGACACAAGGAGGGCTGTTGGGGGGAGGGAGAGGAGTAGAGACATAC
This mutation is a 2053 bp deletion beginning at Chromosome 17 position 34,895,103 bp and ending after 34,897,155 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51306 | CTG GGG AAG TGG GAA AGT AGA | Wild type Forward | A | |||
| 51307 | Fluorophore-1 | CAA GGC TCA CCG GAA ATG AAA T | Quencher-1 | WT Probe | ||
| 51759 | CCC TCC GCT ACT CAC TGT TC | Common | A | |||
| 51760 | GAA CCA CGC CTC CCT CCT | Mutant Forward | A | |||
| 51929 | Fluorophore-2 | AGG GAC CGC GCC TCC TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51306 | 0.40 uM |
| 51759 | 0.40 uM |
| 51760 | 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.