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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
>chr10:31506063-31506185 123bp CCCAGTATGCATTTCTTAAAGC AAAAGTCTTATGAGCATCCCTGAAG
Mut = 127 bp
Wt = 123 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CATACCACTAACTCTGTGTACCTGAGGAGAAAAGAGTAGAGTCACAAGTGAAATTGTAGATTCTGGCAACAACTTACCTGCTAAGCATCAAACAAACTCTGGCAGGTTTGACTCTTTGTTTTAATTATAAAATTCCTAACTACATTAAAATATTCCCCAGTATGCATTTCTTAAAGCCATGTTacaggatcataatggtaaaactaatattgggaaccttataaatacgattataaacaaattgaaaacttcacttcagggatgctcataagacttttaatttggtaaaaccgatctattttacttgacatattttactttttaagcacattaattgcatttccttctatttgtccctttatccagatccatatccagagaacagaagggtgtgaccatatgacttgttcacagtgtaacactaatttttgctatcgatgtggagagagataccgccagctccggtttttcggagaccacacatcaaacctcagtatatttggatgcaaatatcgctacctcccagagagacctcatttaagaagattagttcgagggtcagtctgtggtaagtagcacacatgattgctctcaggtgcctccttaggatccgaggaagggcaatagcatgagaatatcagccatgcgaatctccagggcttgctatgggccaggggttccagtaggtcctttaccaaagagtgagaagagaatcactccttttatgaacaaaattgaaaagttaaggttgttaacatgacattctttcatgatcagcgcttctgaaaccctgggaaagccaattgctgacaagaagaatgtatggacttaacaggtatctaaacacaaggatgtgctgttgattgtttatagaatatgaaatgggacaatgatgaaaaagcacacacatcccaaagaaataaacaaataaaacagtaaatgttaagacacaatcaatggtagattgtcatagcagaaattaaacaaaattttaaattaaaaatgtttaaataagatataatttatggttgtgaaaaattatatttgattattgaaaatgatgttctaagagaattgttactgatcttggaataagagaaagcgtgcttactccactcaaaatcatctttagcagtccatgtcctacactgtcaagtttattccttctaaagggcagatatttgctccttgtattccgtgatggttgggaagaaaatgtcagtgctattattacttgacagttatattctgatgctgtgaagaaatgtcttggtggatactttaaggatatattactaaagagattaagctgtatgtttataatatgtaaccctttgtactgtattctagcttcttccacacagctctatggatacacatgcccttgttcaataagatttcatgtcacttaactgagttttgttattgtgtaaagcaaaagaaaaaaacgtatgtctgggcattatgttaaatggacaacaattcatatctaaatactaggtattaagtaatgctaaaattgatatttaaaactgtatttttttatatctagtcatctaattccaaaactgttttctatgaattaaaaaagttaatttttcctgccaaactcccaacaaatggctgaattatctttcattcattggtcaatgatcataacttatagatggtattgctgtgtgattcttaagtagctgtgtgactactgcagacttcctgttcccttcacactctttgtgacatttcgtttgtacggttatgtcaaaagtcacagcacagtaacaagaaataagtaactgtcctggttttgtctatgttggagccttttagagatatagggagtcctctagtggcttctgggctgcagttacatgtaacaatggtgattattatcacagatcaaatattcattcagcagagaaaccgctggtgctgccaagcgtgctgtgcctgccagcactgagctgtcttacaatgctgcctgttaacatttgtttcagtgattcacttgggtaacttccagcacatcacttacgcagtgggaagttcccctagcagttgtcttcaggcatcccatttggagttcagtctgttagacattcatatgatgtccctctttatctagcaagtaaataaagcattgttcttagacatggggctggtcagacaccctctctgatccctactggctgtctaactttgtgagctcaatagattatcctctgagtcactccattcttaatcgttcaacattttgtacgttgaaagtaggagaaatctaaaggaaaagcacagagactgaagcgcatcagtgaaaaggattatagtctgttttgggtataaatagtaagggacctgtggctacacggcatctgcccatggctttgtggcactgtacatacaccatgtatcagttctatatgtgcccttaattgttcttatgcttctttaaggattactgtaactgctctgtaactcagcagtgagagcctttaactcttatacttttgtttttcagctggaaagctcttcattgcgcctctcatcctggttttgggattggcactaggggccatagcagttgtaatcggtaagacacgtgacatatttgagattagatttctcccacagcagtagagaccgtgtgagtgtacatgtggtggtgtgagtcagtcagtttgtgtggtttctgctctgccttgcttaaaacagccatgcagttcagggcatataagtcagaggaaCATGTTTCCTACAGGATGTGTTTATGAGACTTTAGTATATAAACATATGTATTAAAGGAGTAGTTTTCAAACAGCATGATAGTCTGTAGTCTGTTGCAGACACACATTGCTAGATTCTACTGTCAGTTTCTAGCACAGTGTGCTGGCTGGACCCATGAGTTTACGTTTCTA
This mutation is a 2551 bp deletion beginning at Chromosome 10 position 31,503,602 bp and ending after 31,506,152 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49293 | CCC AGT ATG CAT TTC TTA AAG C | Common | A | |||
| 49294 | AAA AGT CTT ATG AGC ATC CCT GAA G | Wild type Reverse | A | |||
| 49296 | Fluorophore-1 | CAG GAT CAT AAT GGT AAA ACT AAT ATT GGG | Quencher-1 | WT Probe | ||
| 49297 | Fluorophore-2 | TGT TCA TGT TTC CTA CAG GAT GTG TTT AT | Quencher-2 | MUT Probe | ||
| 50767 | CTG CAA CAG ACT ACA GAC TAT CAT GC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49293 | 0.40 uM |
| 49294 | 0.40 uM |
| 50767 | 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.