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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
>chr10:85927489+85927598 110bp AGGAAAATACTTCGTGCTATGG GTTATTTCTCGGAGTGCTTACTGT
Mut= 97 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AAGCCTTCAAAGCCAAATCCTTTCTCCTTGGGATTGGGGCTCTCTTCTTTTCAAATGTTGTTTTCCAGTCCTCTGGGGAGGAAATAAACTTAATGATGAGTACAATAGGAAAATACTTCGTGCTATGGGAAGATATTGTTATATCCTGAGTGggattagaagaggttgggaggcaggactaaaggtgtgtttacagtaagcactccgagaaataactggctaaaagtgtttgttactgttcgggcttttgatgtgtttttacagagcagcagaagcacaaagggccccgcgttaaacataccttagaacacaatgtgcggatccttacattcgatgctaaagtacatatattttgttcttgattcctttgaaaagtgtctaatttggtttttaaagattctatttcgcagaagaaaacctcgggcttccccggaggtccaaggaggacttgacacatcgtctaaccacaagagagcagtcttccctttgatgagcgcttcttctccacctccccagaaatgaaagagcagagaggtatgtttgcaaacactctgaaagctctttccatcagacctgaacgccggaaaccacaaagggcagtcaagtttggattttcaaggagaacgccggagtaaactttcgctctgttggccagccgatcagccagtgcctggggaggctctctgctcaattcctgaacaagttcgggctcccggatagccacacttgctgacccaaggagtgaattacaacaattgggcagctttgaaagatcaatctgcttagagtgcgaccgtctatccaggctgggaaccctatgttaactcttaaactaagatcagaggaagcacatggatacaagcgttgatgaggcgagccctctatctttgccccttcttctctgaagctttataaagacccaagaagagcaacctgcttcggaaatcatgcaaagttcactccaagtgtgctcaggtacctaacttctggacctagaaacttctccagagacagaccgggaagtctggatggaccagaatctccgctacgacagcttccagtctcgtcttcaaggtgagagagtcatctgcgtgctttttcgcagagaatggaaaaacgcagatcggccggactgcttgccctgccgtccgcgctacgtacagcgccgctgggcgcgctgcccgggagggagccctgcagggtctcggtgcgccaggatgccgcggactgcgcgcggagacggccgtactcgtccctgccgcttgggggcgtcgccgagcccgcctttttgcgccaacgcaacgagcgcgagcggcagcgcgtgcgctgtgtgaacgagggctacgcgcgcctccgccagcacctgccgcgcgagctggcgggccagcggctcagcaaggtggagacgctgcgcgccgccatcagctacatcaagcaactgcaggagctgctggagcgccaccggccggactgcaacagcgatggcgagtccaaagcatcctccggagcctcgccctgcagcgagtctgaggagcgtagctagcagcatcctagcccactgggctttcatggttgcttctccgggcctagatctttaaaacaaaaccaaaaaccagaaaacaactaagtgcttctggatctaggaaagaaaggtaattgaatgtttagagagtttggttttttttttttttttttttttttgtcccctttcaccttcattggttccgagtttgcagcttaaaaatgcttaaggaaactcaattctcgtatttttgaagcacagaagggccatctcaaatctatgcggtgattttaaaaacacaaaggactttaagaaaccttagccagagaagaaacacacacacacacacacacacacacacacacacacgagacagtccttcacgcgagaggaacattctcactcattccttccaattgggaaatgcactggaaatctcaacacctcactgagggtaggacactccataagacatttgtggagcatctgctggacctgaacgtcgctgtgctaaaaaggacaatgcttgacctatcttgcttcgtccgcaagaggtagatcggaaactttagcagctaataaagtgccgcgaagttaatgatctttttggaggagctctgaatggccatcttctctcccggtttctgcccactgtttctcttgcccttttgtctatgtgttcccctgtacagttctcatcccgtctaacctttgcttggaaaaaataaaaaagcaagtatgcttagtgccttttggtgtttgacactgacttgacaaatggaaagcaaatatgcacggtgccttttgGCATTTGAGCTGAATGGAGAGAGCTATGGACGATGGGGTTGGGTTGAACTTGCTACATGTCCGAGCCATTTGTCTTTGAAAGCTTTCTAGCGTGTTGAATATTTAACTATGGTCATTCCTTCTACCTCACCAAAACAGTTATGAATTCTGTAAAACCACTAGATGCTTGTTGACAT
This mutation is a 2177 bp deletion beginning at Chromosome 10 position 85,927,535 bp and ending after 85,929,711 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44815 | AGG AAA ATA CTT CGT GCT ATG G | Common | A | |||
| 44816 | GTT ATT TCT CGG AGT GCT TAC TGT | Wild type Reverse | A | |||
| 44817 | AAG TTC AAC CCA ACC CCA TC | Mutant Reverse | A | |||
| 44818 | Fluorophore-1 | AGA GGT TGG GAG GCA GGA | Quencher-1 | WT Probe | ||
| 44819 | Fluorophore-2 | CTG AGT GGC ATT TGA GCT GA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44815 | 0.40 uM |
| 44816 | 0.40 uM |
| 44817 | 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.