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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
>chr17:5418581-5418683 103bp CACTTCCTCCACCTGAGTGC ATGCCAAAGCCCTTGAAATC
Mut = 103 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GTGGCCTCAACTGTCTTCCTCACCCTGTTCACTTCCTCCACCTGAGTGCGCAGCCATGGCCTgggctacaagcatcttgagagccagcagaagagtgagtacaaccagggtagatttcaagggctttggcatagctctatggcccagtcaggtgcaacgtgtccctcccaaccgggcctggtggagcctcacgcccacgtcctccatggcgaccgtgaagggtgaacttatccagcacatcagctccgaagagcccgttctccacaacaaggtctccattgtaggaactggatcagtcggcatggcctgtgccatcggcatcatagcgaaaggcttgactgacgagctcgcccttgttgataataatgaagaaaaaatgaagggtgagacgatggatcttcagcacggcagtgtcttcatgaaaatgccaaatattgtttgcagcaaggatttccgtgtcactgccaactctgaggtagtgattatcacagcgggtgcacgccaagagaaaaatgagacacgcctgaatttagtccagcgaaacgtaaccatctttaaggcaatggttgcggaaattatcaagcacagccctcgctgcaaaattattgttgtctccaatccagtggatatcttaactttcgtaacctggaagttgagtgggtttcccaaaaaccgtattattggaagcggctgtaacctagatacagctcgcttccgttacatgctcgggcagaggcttgggatccactctgaaagctgccacgggtgggtccttggagagcatggagattcaagtgtccctgtgtggagtggtgtgaacatcgccggcgtgccgctgagagagctgaattcagctataggaacaagcaaagaccccgagaaatggggcgatgtccacaaagaagtcattgccagtgcctataatattatcaaaatgaaaggttatacttcctgggccattggcctgtctgtaactgatattgcagagagtatcctgaagaatctcaggaagacgcatccagttaccaccaaaatccaaggtctctacggaatcaaagaggaggtgttcctcagcgtgccttgtatcctgggagagagtggcatatctgatatcataaaggtaaagctgagccctacagaggaggcccagatggtaaagagcgcagagactattTGGAAAATCCAGAAAGATATTAAGTTTTAAGTCACCCTTCTAAAGATACCGAAGACAGAAAACGCAGTGCACGGCCAAGTGTGGGGTGAAGCGGGGGTTCCG
This mutation is a 1112 bp deletion beginning at Chromosome 17 position 5,417,539 bp and ending after 5,418,650 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52227 | CAC TTC CTC CAC CTG AGT GC | Common | A | |||
| 52228 | ATG CCA AAG CCC TTG AAA TC | Wild type Reverse | A | |||
| 52229 | GCA CTG CGT TTT CTG TCT TC | Mutant Reverse | A | |||
| 52230 | Fluorophore-1 | CAT CTT GAG AGC CAG CAG AAG A | Quencher-1 | WT Probe | ||
| 52231 | Fluorophore-2 | CCT TGG AAA ATC CAG AAA GAT ATT AAG TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52227 | 0.40 uM |
| 52228 | 0.40 uM |
| 52229 | 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.