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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
>chr8:47710961-47711122 162bp CTCAAGAAGAAGGTGGTGGTG GGATTCAGTGCCTACTTGGA
Mut= 174 bp
Wt= 162 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GCCTATCCTGGAACTTGCTCTATAGACCAGGCTGACCTCAACCTCAGGGGTCATGCCTGCCTCTGCTTCCTGAATGCTGGGATGAAAGGCACTTTGCTGCCACCCCACTCTCTGTGATTTTTAAATGAATGTTTTGTTGATTGTCATGTCTTTAGGACTCTTAGTAAAATACCTGTGTTTTTCTTCTTTCTAGAAGGGGTAGAGGAGCCATCcaaaaaacgagccgtagaaggaaaaaacaattcttcagttgagcgagatcatgggaaaaaatctgccaaaacggatcgttcagcagctcagcaagaaaacagttcgccgtccagggggtcgtccaccaaatccgagagtggtgggacctcagctcggagcagctccagtggctcccatcaggatagcgccacaagtgaaggagacagatctgtgtgcagccagagcagcagcaattcctctcaggtaaccgcagggtctggaaaggctttggagtcagaagctccacataaacgtggttcagcgtcatttgtttcttccctgttgaagtccagtatgaatagtcatatgacccagtccactgataacagacaacagagtgggtcacctaaaaagggtgctctggaaggttcttcaggctcagcctctcagagcagctcagagattgaggtgcccttgttgggctcctctggaagtgcagaagtagagttgccattactgtcttgtaagtctagttcagagacagcttccagtgggttaacatctaaatctagttcagaggcaaacatttcatcatcagtttcaaaaaacagttcatcgtcaggcagctctttactaaccccccagagcagttccacaaatccatcgctgctgacatccaaaagcacggctcaggtagctgcttcactgctagcgaccaaaagtggcgcttccttaggaagcgtgtctcagctagctgccaagagtggttctcagagtagcacttcacagttgccttcgaaaagtacctcccaggccagtgagagttctgtcaagtttgcttgtcggaagttaaccaatgaagacataaaacagaagcaacctttcttcaatagactgtataagacggtggcgtggaagttagtagcagttggtggcttcagccccactgtgaatcacggagagctgctgaacgcggctattgaggctctgaaagcaacactggatgtgttttttgtcccactgaaagaactggcagatctgcctcaaaataagagctctcaagaaagtattgtctgtgaattgaggtgtaagtcggtatatttgggcactggctgtggaaaaagcaaagaaaacgcaaaagccgtcgcatcgagagaagcactgaagttatttctcaagaagaaggtggtggtgaagatctgtaagaggaagtaccgaggcagcgagatcgaggatctagtactcctggatgaagaagcaagacccgtaaacttacctccagCACTGAAACATCCTCAAGAATTACTGTAACTTCTCCAAGTAGGCACTGAATCCAGTATCTGGTGTTTGAAGAGAAAAACTGGCTTTTATATAAGACACTGGCTTTCACAGATTTTATATTGCTTTTTTCCAGTTTTGCAGAAAATTTAAATTCTCGTTCTCTTCACTAAAGTAGAGCTGTAAATAATTTATGACAGTGCATATTAAAGGGTGT
This mutation is a 1262 bp deletion beginning at Chromosome 8 position 47,711,014 bp and ending after 47,712,275 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52805 | CTC AAG AAG AAG GTG GTG GTG | Wild type Forward | A | |||
| 52806 | GGA TTC AGT GCC TAC TTG GA | Common | A | |||
| 52807 | CTT TGC TGC CAC CCC ACT CT | Mutant Forward | A | |||
| 52808 | Fluorophore-1 | AGA GGA AGT ACC GAG GCA GCG | Quencher-1 | WT Probe | ||
| 52809 | Fluorophore-2 | AAG GGG TAG AGG AGC CAT CCA C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52805 | 0.40 uM |
| 52806 | 0.40 uM |
| 52807 | 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.