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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
>chr2:180714297+180714398 102bp AGTGGATGCAGGAATCTTGG TGAGGCAGACACCTTGCT
Mut = 92 bp
Wt = 102 bp
Fam = Mut
Hex = Wt
WT reverse primer anneals over the nucleotide sequence containing mouse genomic variation rs262999462.
Sequence
Wt Sequence (deletions in lower case):
TCTTAGAAAGTGGATGCAGGAATCTTGGCCTGTGGTTTTACCAGACTCCTGTGgatgaggtggcgccagagcaagcacccctgtgaagatgcagcaaggtgtctgcctcacctgcaattgcctctgttgctctggcatcctgacctgtaatccctgccttgatgatgtcacagcacacaatgggctagcagtctgtgaccttcaggttgataggctaagccttttgtatagctgcaaagaaatgacagccttgactattaatcacaaaaatagtaaaaaccaagttcctaagaaactgtggtttatatgtaggatcatttaatgatggttgggctctttagaagtacaaagccttaattcctagttatctttcctatcagtgaagatagttttcttttcagagggctttaaggaagcagcagagaaatttcggatggagtctgggatcgaaccaagtgtggacctagaaacacttgatgagcgaatcaagattcgggagatgattctgaagggtcagatccaggaggccattgccttgatcaacagcctccacccagagctcctagacacaaaccggtatctttacttccaccttcaggtgagtttcagcagtctgatatggctgctagacagacaaattaagcttgtttgcagtggctcagaaagaaatcggccattgtgtgtaaggagacaggctttgagctagctgctgctttatgtactattgcaactagttactttttaaaggggggtgtgtactggtaataagaacacatgttccaccatgttcatagcagccttatttataatagccagaagctggaaagaacccagatgtccctcaacagaagaatggattcagaaaatgtggtacatttacacaatggagtactattcagctattaaaaacaatgaatttatgaaatttttggacaaatggatgtatctggaggatatcatccttagtgaggtaacccaatcacaaaagaagtcattagatatgcattcactgggggctggtgagatggctcagtgggtaagagcacccgactgctcttccgaaggtccagagttcaaatcccagcaaccacatggtggctcataaccatccgtaacgagatctgatgccctcttctggtatatctgaagacagctacagtgtacttacatataataataaataaatctttaaaaaaaaaaaaaaaaaaaagatatgcattcactgataagtggatatttgcccagaaacatagaacccccaagatacaatttgcaaaacacaagaaaatcaagaagagggaagaccaatgggccgatacttcattcctccttagaacagggaacaaaatacccatgaaatgagttacagagacaaagtttggagctaagaagaaaggatggactatccagagactgccccacccagggatccatcccataatcagccaccaaacccagacactattgcatatgccagcaagattttgctgaagggaccctgttatagctgtatgaggctatgccagtgcttggcaaatacagaagtggatgcccacagtcatctataagatggaacacggggcccccaatggagaagctagagaaagcacccaaagagctaaaaggggtctgcaaccctataggtggaacaatatgaactaaccagtacccccagagcttgtatctctagctgcatatgtagcagaagatggcctagtcggccatcactgggaagagaggccccttggtattgcaaactttatatgccccagtacaggggaatgccagggccaagaagcaggagtgggtgggtaggggggcagggtgggggggagggtatagggaactttcaggatagcatttgaaatgtatataaagaaaatatctaacaatcaatcaataaataaatggataataaaaactatacttttatcaaggaaaaaattaaaggggtgtatgtgtctgtgtgtgtgtgtgtgagagagagagagagagagagagagagtatgaatgaatagtgtacatatggaaataagaggcattccttgggagtccattctttctgcccttacatgggctctgggcatacttgcacaacgagcactttcactgtggcagtctcaccaatctctccttttgcttttaaaatggaaatgtgccaggcgtgatggcgcacacctttaatcccagcactcgggaggcagaggcaggtggatttctgagttcgaggtcagccagggctacacagagaaaccctgtctcaggggaaaaaaaaatggaaatgcatcttggagatgctaagaaagtttcttttaagatgcctttcttattttcttttagggtgatggggtggagcccaggcattgcctagatgctaaacaagtactttaacactattagttagacactcaatccacacataagatatacttttgattggagccaagtgttgatggttattcatctaagtgtatgccagccaaacttggttgtctttgctgttgaccatactgggttccacctactgggtgtacaggctggctaatcttgtttgtctttgttttgactagcaacaacacttgattgagcttatccgtcagcgtgagacagaagcagcattggagtttgcccagacacaactggcagagcagggtgaggaaagccgagaatgcctcacagagatggagcgcacactggccttgctggccttcgatagccctgaggagtcgccttttggagacctccttcacatgatgcagaggcaaaaggtaaagggccagtaaaggaagaacagagtgcttagacactcagaggacttgtgaaaagatggtactgtgctgcgcttgtgttggggtgaagcacatggctctgggccaggatactccagtagaacactggcagctccactgatctgtccccttttaccttgtgagagGAAGAGCAGCGCCATTGTGACTGTTAGTGTCTAAAACCCT
This mutation is a 2882 bp deletion beginning at Chromosome 2 position 180,714,342 bp and ending after 180,717,223 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53056 | AGT GGA TGC AGG AAT CTT GG | Common | A | |||
| 53057 | TGA GGC AGA CAC CTT GCT | Wild type Reverse | A | |||
| 53058 | GTG GCA AAG GGT TTT AGA CA | Mutant Reverse | A | |||
| 53059 | Fluorophore-1 | CCA GAG CAA GCA CCC CTG | Quencher-1 | WT Probe | ||
| 53060 | Fluorophore-2 | CCT GTG GAA GAG CAG CGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53056 | 0.40 uM |
| 53057 | 0.40 uM |
| 53058 | 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.