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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
>chrX:135843474+135843576 103bp GGAAGAGCAGGACTCTGTGG AAGATGCTCGCGAATTTCC
Mut = 108 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TGGGAAGGTACAATACTGTGACAGACCTGTCATTGAGACTGAGGCTTTGGTAGGCATTCTTAAATTGAGCCTTCTAACTTGTACCATGACTGGGGCAGAAGTTGagaccggttctcaggccaagcctgataagaagcctcaggaagaggtcgcaggtggggctgagagagaaagtgaagcccctctggtggtcagacccaaagtcaggcctcaggctccagcaacaagtggtgctaggcccaaaactgaaactaaatcttcctctcgggcaaggcctaaaactgaaacccagtcagtgtctggaacaagacaccgaatgagtggagcaaggccgagatctgaggctcaattaatgtctggggccaggcctaaaaccgatgccagggcagtaggtggtgctcgccctaagactgaggccaagccaatcccaggagcaaggcctaagggtgatgcccaggcttgggcccacagtgagtttggagccgaggcaatgccacgtgctgagagagcacacttatctaattctgtcacatggccaccagtcaatgttgggtctgcaactgttactaaatctaagagtctgtctatgaatacagaactggccagtatgggtagtgaaatcttttctggcacccagggtcagcctggaatcgagccctggtttggaccgagagaagaggctaatatgggttcttggtgctatcccaggcccagagccagagaggagacctctaatgagtctgcagatgaaaactctacaatgtcttctttctggactagagaggagactagtatcagatcatggcccagggaagaggtgaacactaggtccaggcacagggctaaacatcagactaatgccaggtcgaagcccagatccaaacaagatccctatattgattctttgtctgggtccgaggatgaggccagtaacccattctgcttttgggctggagaaaatactaatgacatgtttagggccagaggcagggatgaggcgaatgccagacccaagatcaggacaaagagagaggactattttgaagatgaagatgagatctataaagagtcctggcttttgcctggggaagagggtaatagatttaggcgccgagacaaggaagagcctaataagaccttgaaaaatgagaacgaaaaagatgttaaaaatgatgaaacagtcgaacaagagtccaggcttgaggaggaagtcattattgggtcctggttctgggcagaacaggagactaatgtggaggctggagcttcagcaatctgtgacgctgaaccaggggctgaagagggggccattggtggatccctgttctggaccgaagaaaagcctagtttgggggctgtggccagagatgaggtcaggcctgagtctgaagaagaggcactatttgggtcctggttttgggatagggatgaggcctgctttgacccaaatcctactcctgtgtacacagctaagagcaggtacagagatccagaagaggatcttaatttagcatccaggcccaaaacctgggacgaggttaccatcgaattcaaacctccttgccatgggcttgggttcccttccccaagaccctttatcattcctgaaggggcttctggaaatagtgaggaaaaagccaagaatgcagagcttggcgcagagggggaagagcaggactctgtggctcagcgtgatcttcctgaacccgagtttccatttcagtatgatccctcttaccgctcagtccaggaaattcgcgagcatcttaaggccagggaaagtgcacagccagagaattggtcttgcacctgcatccagtgtgagcttagaattagttctgcagaatttgaggagcttcttttactgatggacaggattcgtgatccttttatccatgagatagctaagattgcaatgggcatgagaactgcttctcagtttacccgggatttcattcgcgattcgggtgttgtctcactgattgaagccttgatgaattatccttcctcccgagtgaggactaattttttggaaaacatggttcacatggctcccccttatccaaatctaaacatgattgagacatttatttgtcaagtgtgtgaggagacactttcacatagcgtgaattcccctgaacagctaactggaatgaggatgcttagacatctcactataactactgactatcacgtactgattgccaattacgtgtctgggtttctcgccttattaaccacaggcgatgcaagaaccaagtttcatgttctgaaaatgctgttgaacttgtctgacaatccgatggtggcaaaaaaactattcagtgccaaagctctatcaatatttgtgggtctctttaacatagaggagactaatgataacattcagattgttattaaaatgtttcagaacatcagtaatattgtaaaaagtggagcgatgtctttacttgatgatgatttcagtcttgagccgcttgtttctgcatttcatgaatttgaagagctagctaaacagctgcagatccaaatagacaatcaaaatgatcccgaGGAGGGACAGTAAAGTTAGTGTGATTTAACCACCTGCCACTCATCAGCCTTATGTTCCCAAAGAGTCCCAAATAGTGCTTTGGTGTTC
This mutation is a 2449 bp deletion beginning at Chromosome X position 135,841,913 bp and ending after 135,844,361 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51466 | GGA AGA GCA GGA CTC TGT GG | Wild type Forward | A | |||
| 51467 | AAG ATG CTC GCG AAT TTC C | Wild type Reverse | A | |||
| 51468 | TGA GGC TTT GGT AGG CAT TC | Mutant Forward | A | |||
| 51469 | TGA GTG GCA GGT GGT TAA ATC | Mutant Reverse | A | |||
| 51470 | Fluorophore-1 | CTT CCT GAA CCC GAG TTT CC | Quencher-1 | WT Probe | ||
| 51471 | Fluorophore-2 | ACT GGG GCA GAA GTT GGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51466 | 0.40 uM |
| 51467 | 0.40 uM |
| 51468 | 0.40 uM |
| 51469 | 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.