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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
>chr7:98716402+98716500 99bp TGAACAGCACCTCAAAGCTC TACATGTCAGCATGGTGCTCT
Mut = 95 bp
Wt = 99 bp
Fam = Mut
Hex = Wt
Sequence
WT Sequence (deletions in lower case):
TTTGTGGATTTCATTTCTATCCCTGTAACTGTCTGTATCTGTGGCCTTGGGCAAGTTATTTACAGTCCTTATTTGTCAGAGGGATTGATACCATCTTCCTGAAGAAAgtaacgtgatccccgtaaagtgctggccacagtgcctgtaggaatggttgttctcctcaccttcctttttctcatggcatggattttgtttattaaaatctgtctgcagcatttgtccctttctaacttatttttgtgtcctactttgttaatgttctattagtggtgaatgaatgcatttgtggattttaaatatttagccaatcaaaagtaatttgttttgtcgtttgtggttcttcatgagtctgtttataaaagtaactttttgttttccagttgaggagacttctgaacaagagcaggaaactaacaccaatgctcagaaccccagtgcagaagcggtgaaccagcaggatgcaaatgtgctgcctttaacccttgaagagaaggagaacaaagagtacctcaagtctttatttgagattctggttctcatgggaaagcagaacatccccttggatgggcacgaggctgacgaggtccccgagggcctctttgccccagacaacttccaggcgctcctggagtgccgcatcaactctggcgaggaggtcctaaggaagcgctttgaggccacagcggtcaacacattattctgctctaaaactcagcagaggcacatgctggagatctgtgagagctgcattcgggaggagactctcagggaagtgagggactcacacttcttttccattatcacagacgatgtggtggacatagcaggagaagagcacctgcccgtgctggtgaggtttgtcgatgacgcccataacctgagagaggagtttgtgggatttctgccgtatgaagctgatgcggagattttggctgtgaaatttcacactacaataactgagaaatggggattgaatatggagtattgtcgtggtcaggcttacattgtgtccagtggattttcttccaaaatgaaagttgttgcttcaagacttttagaaaaatatccccaggctgtctataccctctgttcttcctgtgccttaaatgcatggttggcaaagtctgtccctgtgatcggggtgtccgtggctttaggaaccattgaggaggtctgttcttttttccatcgatcaccacagctgcttttagaacttgacagtgtaatttctgttctttttcagaatagtgaagaacgggctaaagaactgaaggaaatttgtcattcccagtggacaggcaggcatgatgcctttgaaatcttagtggatctcctacaagcacttgtcttatgtttagatggtataataaatagtgacacaaatgttaggtggaataactatatagctgggcgagcatttgtcctctgtagtgcagtgacagactttgatttcattgttaccattgttgttcttaaaaatgtgttatcttttacaagagcctttgggaagaacctccagggacagacctctgacgtcttctttgcagccagcagcctgactgcagtactgcattcacttaatgaggtcatggaaaatattgaagtttatcatgagttttggtttgaggaagccacaaatttagcaaccaaacttgacattcaaatgaaactcccagggaagttccggagagcccagcaaggtaacctggaatctcagctaacctctgagagttattataaagacactctcagtgttccaacagtagagcacattattcaggaacttaaagatattttctctgaacagcacctcaaagctctgaaatgcctatctctggtaccctcagtgatggggcagctcaaatttaacacatcagaagagcaccatgctgacatgtacagaagtgacctacccaaccctgacacgctctctgccgagcttcactgttggagaatcaagtggaaacacagagggaaagatatcgagctcccgtccaccatttatgaagccctccatcttcctgacatcaagtttttccctaatgtatatgctttgctgaaggtcctgtgtattctccctgtgatgaaagttgagaatgagcgctatgaaaatggacggaagcgtctcaaagcatacctgcggaacactctgacagaccagaggtcaagcaatttggctttgcttaacataaattttgatataaaacatgatttagatttaatggtggacacatacatcaaactctatacaaataagtcagagcttcctacaattaattcagaaaccattgaaaatacctagaagattttaaaaatggctgttatcttattttgctgtttggaagaaaagctgtaaggtatagtaggccagataatgacttgctgatagacctccagtttaacatactattagctattggtaatccacgtgtttaaatggcccctgtttgaacactcatgctttcaatacctgtctgttcttccagaagttggcattaggagtgccaaaatacctctgctggtggcactctgaaaatgttttacttaaaagtcgttttaggggctggagaaatggctcagcggttaagagcactgactgcctttccagaggtcctgagttcaagtcccagtgaccacatggtggctcacaaccatctgtaatgggatccgatgccctcttctggtgtgtctgaagagagcaacagtgtactcatatacgtaaaataaataaatctcttaaaaagtcattttagatatgacattatcactgtcgatccagttgtcagttattaagttatcaggtctttggaaaatgaattttgtgagagaaatacattttataatgtgtcatgatgaaaaacaccattgactttgacttggagtttcaggtgtttccgagctgtggtagcagcacttgggaggcagaggcatgaggatgacaggctttaggcctggaccacatagtgagcctgcttcaaagaacgagtgagggctgactggggtggagcctagtgtggagctcttgcctcagtactcaaaagcaaaaagaacagcaaaagtcacaggaagtaacgagcagccttctgtgtagatcctgtcagggcagaagaacctcaaaggaacaagttacagtagcagagccagccctgctgctcctcacttgattgctgctgtttgcatttctttgctgagcccacattttcataggcttgtttgctattcacttgtgcttcatggagaagaaagacccagaggccatccctataggcagctgacttgtctgttgtctccttacatgactgtgtctgctgccttggctcggttataagcaaaacctacagaaataaatgttttgtggtttatctaaaaataatacagaaaatattgctgttatttttggtgaataaaatcaattttgtatagtttatttcaacctaaataaaatgtgaattttgtttaaatttcaggtgaggtgtttttggtgggtcagaatacatccttgtatattttcttaaaATGGTTATTTCATAATCAGCCTCATGCCTTTTACGATTCAAGATGAAGTATTTTCTAGCTTTTCTCTT
This mutation is a 3412 bp deletion beginning at Chromosome 7 position 98,714,706 bp and ending after 98,718,117 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 54669 | TGA ACA GCA CCT CAA AGC TC | Wild type Forward | A | |||
| 54670 | TAC ATG TCA GCA TGG TGC TCT | Wild type Reverse | A | |||
| 54671 | GGC CTT GGG CAA GTT ATT TAC | Mutant Forward | A | |||
| 54672 | AAG GCA TGA GGC TGA TTA TGA | Mutant Reverse | A | |||
| 54674 | Fluorophore-1 | ATG CCT ATC TCT GGT ACC CTC AG | Quencher-1 | WT Probe | ||
| 54675 | Fluorophore-2 | CAG AGG GAT TGA TAC CAT CTT CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 54669 | 0.40 uM |
| 54670 | 0.40 uM |
| 54671 | 0.40 uM |
| 54672 | 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.