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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
Mutant= 80 bp
Wild Type = 82 bp
chr13:64921512+64921593 82bp GCTTGCCTTTCTCAAACCAG TAGACTCCACCTGTGCTTTGC
Sequence
Wt Sequence (deletions in lower case):
ATAATAACCTTATGCCCTACCTTCCACCATCATAACCCACTCTCCTAATTTCCAGCTtggggagaatgcctgaaaaagctgaaagaaaaagaaaaggataaattatttctagaagaaatgagcccaggacaccatttgaactctttagggaatatatttaactcatcaagtgctaaacaagacagcactaccctgtcacccttttggaacctgaaagaaaaatcagaagagcaggtggccactcagaaactatcttatcccaagatctcagaagaccattttcaacagaaatgtgaccagcttttctggggtctcccttctctccacagtgagtctctggtagctgccgcctggatccctcagacaacgtcaactctcccttctccctttttcttattcaatgtcatctcaagtgtttatccaattcaactgcaggataaaatgtcaccaatgcttccccatacacatcctctgtcctatctggacctccaatctccacacttaattctgtctccacttgaattccagaccccagctttgaatctcccagtcctactaccctcttctctaccctataatagtgattttggtacatcttattctcaatcacaaagtaagccacaatacctcccaactgaaataaaatacaataaaaaaccctcactggcaaaacaaatagaaaatagatcaactttacccttgatggtccagaaacctcaagaagcctatgacatcttggcccccaacccttcccaagactgggtagtctccatccttcctgataattttcccatctgccgtgagctccgggagaaactggagcaacacattcaaaagtggctcattcaacaccaatggaattttccccataagatccaagtgtctgagaaaatgaaggagcttcagaacacagtaataggaaattgtcaaaccagagacaagcctggcaccttacaagcccttggtgaacacagcaatgagggccagaagagaaaattccagctagaaaaggaatctggcaagaatctgggacctattctaggaaagatctcaaaagatccaatcaggggcttggaaaaaactacagcaatcaaagatttagaaaacaacttgaaagctcatttgggtacaaagtcagggcagatagatcaaggtctgacccctctaagtatgcgacaatcctggcttgctgtggatgatagtttttacgagatggaaaacaatttaacatccttaaagagttcagcaaagtccatgtgctcttcagagaagcttgcctttctcaaaccagaaactcgacaggctctggaggcacatattgtaaggttttgggcaaagcacaggtggagtctaccccttaagatactcaagcccgtaaaactctttcagttaagtattgagtccttgcctgttgtactctgtgcccagacctcctcaaccacttctgtacataggaccagatcagcagctgaagtagtcagattcctaggaaaaccatgcttgagacagatgataattgaggattcttctccatcaccacagaatcttctccttgtctcatctccttcctgtaagagggccaggagaaggcttccctttggtgttgaccatgagccttctacagccctgccaaccaaaccagagtgtgggcatatttcagaagatctcacttataatttcatgaacacaacctctcagactaggactatcttcaagaaagagatagagactcgagaagtcgttttactgcctagaaggacgagtgaccaaaatttggaggcatacaaacatcaggaaaaagttgttagtgagtttccacataatgtggagacagaattagcaggccagccacaaatctataccactactgtgctccctccaaaacgttccaggagcatgcctcttcctgtagacactttgacttcacatgtattaggtgacattgtggtggcagacatggacaatagcctggtgcagcagagacccagtactccaaagcatctagtctcacagaagagccaaatcaagatgttggcccctacttatcaaagtgagggcactaagagacagagtgaaataaagtatgaagacagacctaagctcaccccagtcacagagaaaaaagccaactttggcagccaatactaccagacccttccaaaaatcgcacaagtgcttccaggaaggcccccccaaagacatttaggtcgctttctacagtggattcatcctaagaaaacaatcaaagggcatgaatttcatcccctaaaaggtaatcctacagcagccattgtccagaaccaacgaaggcaagtgagaaaaataccatatatggacaacaatgtgactgaggcccaggaacttatgacaactgttggacagatgttagaaaagaaaatgatgctgcaacatcaaccctatgcttcaaaattcaaccagcacagggaagttcctccagcccccatgtctcggttttcccatggccacatgccagtctcctacttacagcaaaggagagcacccagttaccctggcagttgctcatgtcagaggtgttctgtacagaacaggcacatcagaaaccaactgcctcagagaagtgtacgctttagcaagaaaccacagaacccacggaaccccagccacaagaaaccacagaacccacggaaccccagccgcttgtcccgtaacgcagctttgaacctagtgaattctcagaacagaacaatagtgccaggaaattcaaatcaccatctttattgtcctaggcactgtgccctccagagAGATGTCTGTAGAGAACTAGGACATTCCTCTGTAGTTTTTCCTAA
This mutation is a 2729 bp deletion beginning at Chromosome 13 position 64,920,298 bp and ending after 64,923,026 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 68127 | TTA TGC CCT ACC TTC CAC CA | Mutant Forward | A | |||
| 68128 | CAG AGG AAT GTC CTA GTT CTC TAC A | Mutant Reverse | A | |||
| 68129 | GCT TGC CTT TCT CAA ACC AG | Wild type Forward | A | |||
| 68130 | TAG ACT CCA CCT GTG CTT TGC | Wild type Reverse | A | |||
| 68131 | Fluorophore-1 | CTG GAG GCA CAT ATT GTA AGG TTT | Quencher-1 | WT Probe | ||
| 68132 | Fluorophore-2 | CCC ACT CTC CTA ATT TCC AGC TAG ATG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 68127 | 0.40 uM |
| 68128 | 0.40 uM |
| 68129 | 0.40 uM |
| 68130 | 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.