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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
>chr18:32079408+32079525 118bp GGAGTTAAGGCTCCATTTTGA GGCCAGCCTGTGAAATGAAC
Mut= 120 bp
Wt= 118 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TTAAGGGTAGATCCAAACTCTCCAAGTTTTATGCTACACTTCTTTGTTTCTTCTTGAGAAGCATATTCCTGCAGTCCTATAAAGATCCCCCTTCTAAATTGGCTGTAGTGGTTTCTTTGGGTCACCAGAATTATGTGAGGAGTTAAGGCTCCATTTTGAACTACTGAGACCCTCCacatctggaagcatgctatacaggcttgattatacttctctttgggagctagactatgacagttcatttcacaggctggccttaatttaaagtgatgtgtctgtagaggtataggataaaaaattcttttagtattcacaaattcctacatctaaaaaaaattgctgaaaaattttaaagtttcactgccttttttgacattgctatctttttctaaatttttcagaatgaaactagtgatagagaagatggcttggccaagatacataatggaacagactctgagaatgatgaaccttccaatgctaatgccagtgactctgaaagtgaggagcttcacaggccaaaggacagtgactctgactctgaggagcatgcagagtctcctgctagtgattctgaaaatgaacctgtcaaccaacatgggagcgactctgaaaatgaagagctgcttaatgggcatgccagtgactctgaaaaggaagaggttagcaagcatgccgccagtgactctgaagctgaggacactctgcagcctcaggtcagcgagtctgacagtgaagatcccccaaggccccaggccagtgactcagaaaatgaggagcctcccaaacctcggataagtgactcggagagtgaggagctccccaaaccccgagtcagtgactcagaaagtgaggaccctccaaggccccaggccagtgactcagaaagcgaggagctccccaaaccccgagtcagcgactctgaaagtgaggaccctccaaggccccaggccagtgactcagaaagcgaggagctccccaaaccccgagtcagcgactcggaaagtgaggaccctcagaaaggacctgctagtgattctgaagctgaggatgcctccagacacaaagagaagccagactcagatgacagtgatggggagaataagagggaagattctgaagtgcagaatgagtcagatggccatacagacagaaaaggactccacagctctgacagcgaggaggaagaacccaaaaggcaaaaaattgacagtgatgatgatgaagaaaaggagggggatgaggagaaagtagcaaagcggaaggctgctgtgctttctgatagtgaggatgacgcaggcaacgcatgtaagcattcctattggactgtcgctttccttattgactctagctacagctagaatactgggtcctcttctggtatgttttattagcatatattgattgtaatgttagtttcttcatggctatttcacataagtacataatgtattttaatcatacttatctcattatcctgtcttgtccaactccctctCATACTCAGTTTTTACTTTTGTTTTGTAACCAAATGAGTTTTATTAGGTTTACCTTTTGGAAGTTGTGTACTTTTCCAGTGGCTCCACTGCGGAAGAAAATATTTCTCCTTTCCCTACCAACCATTAACTGAGTATAAATCATCAGGGAGGATGGGGCTTCAAAAATTCATCCCTCTACATGGCCATATATTGAT
This mutation is a 1323 bp deletion beginning at Chromosome 18 position 32,079,445 bp and ending after 32,080,767 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45405 | GGA GTT AAG GCT CCA TTT TGA | Common | A | |||
| 45406 | GGC CAG CCT GTG AAA TGA AC | Wild type Reverse | A | |||
| 45407 | GCC ACT GGA AAA GTA CAC AAC | Mutant Reverse | A | |||
| 45408 | Fluorophore-1 | CTG GAA GCA TGC TAT ACA GGC | Quencher-1 | WT Probe | ||
| 45409 | Fluorophore-2 | CCT CCC ATA CTC AGT TTT TAC TTT TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45405 | 0.40 uM |
| 45406 | 0.40 uM |
| 45407 | 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.