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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:122396919-122397029 111bp GGCTAACAGTATGGCAGAAGC TCAGATTCATCCCCAGAATCA
Mut = 114 bp
Wt = 111 bp
Fam = Mut
Hex = Wt
X-Linked
Sequence
Wt Sequence (deletions in lower case):
GTCCTTTTCTGTCCGTCTCTGGGTGCACGATCTCTGCTGTTGTCACCACCTTAAATAGGTGTACGGGGCTAACAGTATGGCAGAAGCGGATCCTAAAATGGTCACAGAACCTGGTGCCcatggggttgctgaagaggcgatggctagcacagcttgtgattctggggatgaatctgacagcaatagctctagcagtaccaatagttgcagcagcagcggcagcagcagcagcggcagcagcagtagcagcagcagcagtagcagcagcagcagcagcagcagcagtagcagtagcggtagcagtggcagcagcagcaatggcagtcatttgaaccgaaagaagagggtacctgagccttccagaagggcccagcgacgtccctccgggaaacttttcttggataagctgccccaagccgtaagaaatcgggtgcaggcactcagaaatattcagaatgagtgtgacaaggtagacaccttgttcttaagggcaattcatgatcttgaaagaaagtatgctgaactcaataagcctctatatgataagcgttttcagatcataaatgcagaatatgagcctacagaggaagaatgtgaatggaattcagaagaagagttcagtggtgatgaagaaatgcaggatgacacacctaatgaaatgccacccttagagggtgaggaggaagaagaaagctgtaatgaaaaagctgaagtgaaggaagaaggaacacatgttccagaagaagttcctgaggcaaaagttgaagaagaggaggctcccaaagaaactcctgaggtgaaaactgaagaaaaagacattccaaaagaaggtgctgaagaaaaagctgaagaacaggaatcctctaaagaaattcctgaggtaaaaggtgaagaaaaagcagactctacggattgtatagatatagctcctgaagaaaaagaagacgtcaaagaagttacccaggcaaatacagaaaataaggatcaacctacagaagaatttacaccaagggctccagcaagagaggctcaaaaaagggtccctgagacaaggcctgaagaaggagtcaatattaaaagggctcgaaagggaaaacctaagaaagaagatcctaaaggtattcctgactactggctgactgttttaaagaatgttgataagcttgggcctatgattcaaaagtgtgatgaacccattttgaagttcttatctgatgtgagcctgaagttctcaaaccctggccagcctattggttacacttttgaatttcatttcctacctaacccatacttcagaaatgagctcctgatgaagacatacataataaggtcaaaaccagatcactacgacccgttcttcgcgtggggatgggaaattgaagagtgtaaaggctgcaaaatagactggagacgaggaaaagatgttacggtgacaaccacccggagtcgccctggtattacaggggaaattgaagtccagccaagagtggttcctaatgcatccttcttcaatttcttcagtcctcctgagattcctttgattgggaagctggaaccaagagaagatgctatccttgatgaggactttgagattggtcaaattttgcatgataatgtcatcttgaagtcaatctattacttcacaggagaaatcaatgatccctactACCATGACTTCAGGGATTATGGAAATAGGAAGTACTACAAGTAGAAAGGGCAAGCTGAAAAATCTTTCAAGAATCCTTAAAAGTTAAGCAAGCAGAAGTGAAATAAGAGTCATCTAAACTTCTAAAATAGAAAACTTGCCCCATAACCTGAACAGTATTACTCCTTATAGTC
This mutation is a 1549 bp deletion beginning at Chromosome X position 122,395,429 bp and ending after 122,396,977 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51259 | GGC TAA CAG TAT GGC AGA AGC | Common | A | |||
| 51260 | TCA GAT TCA TCC CCA GAA TCA | Wild type Reverse | A | |||
| 51261 | TTT TTC AGC TTG CCC TTT CTA C | Mutant Reverse | A | |||
| 51262 | Fluorophore-1 | CTG AAG AGG CGA TGG CTA GC | Quencher-1 | WT Probe | ||
| 51263 | Fluorophore-2 | CCA CCA TGA CTT CAG GGA TTA TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51259 | 0.40 uM |
| 51260 | 0.40 uM |
| 51261 | 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.