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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= 89 bp
Wild Type = 87 bp
Fam=Mut
Hex=Wt
>chrX:45090790+45090876 87bp GTTCCCTGTTGTCTGCCTTC CCAACTGCATTATTCCATACTCAGA
The Mut Probe (62691) anneals over the nucleotide sequence containing mouse genomic variation rs264564392.
Sequence
Wt Sequence (deletions in lower case):
ACAAAAGATAATGCTTCGTCAAACAAAAATAAGAGAAGCTTAAAAATGATAAACAATAATAAGAACTTTAAAAGAGAGAAACAAATCATGCCTATATGACTTTTAAACCCTGTTTGAAATATTTTCTCATTCCAACTAGTCTGTAGCAGAGTTGATTTAGGAAGTGTCTTTTGTGTCCAACTGTTATTCTGTCAAAAGCAGTTCCACATTTAGCTTTGAAGGAGTATACAGATTTATCCCTTCCCTGTTCCCTGTTGTCTGCCTTCCTCACTCCTTTGCctagcaactgatgacttcactaatagaactctgagtatggaataatgcagttggttcctgccttcattccagttgccaaacctctctgaggcagaagcaaagatcacttctccagactactcggctggtgctggtgttcagctggtttgctcttccatctcatgaaagtaatctttatcctatttctacatggcttgtactgaaaatgggtaagtataaaactgagttccactcagttaaatttgaaggcaaaagattattttgttgttattaatgtgtttactatggcaaggacaagaggatcagaaggtaaaagttaaagaaatgtaattgtagataatattatgaattgtaagcatcaaaaacattaacgattgtaaatgtaagattgaaatgaaagttacaaagccctaaacattagggctatatcatggggaaattgttgctctaatagatcttgatgtagattaaaagaagaaattttaataaagtttgtttagaatatgtgatgaatcataaagaaagaagcagaatgtttaaagagaatgactaatcttcctttgacttctgtaatagaatgtaatgctgagttcctgtgccaactccccagagacttctcataacattcctgccagaatcagaattctgagcttagtgggttatagatcttgtaaggacttgacagagctgttactttctgtcaactgggaaggaaataggcatttttactacctttgattgataaggaattaaacaggcacagagggcagccattatgggagatcatgtactaaaagtgacttctgactttgtttgttggtgagggaggggagtgtcagaaggctaggaaaatgtttatatgactatatggagggaaaacctatactcctttatttcctatgtttgtgaaatacaatcacatgctagaaatatttgctgggtaatgtatttccattttatttgaattttcatttctgtttttgttaggcttgcagggcatagccattcacccataatagttcctgtggataaaaacagaagcaaggagacatgccacaatgtgcagctccgctctctgagttccatgttgaaggatgatgaggatgatgcagatgtctggacccgccctcctgtctccttgagacctcccttcaatgtgccaagaacaggagcccgcattcctcaaaatccaagagctccaagacaccctcttacactaacacctgctattgaggaagagtccctagcaacagcagaaataaacagctcagagggcctggagagccagagccagaagggacatgattctattaatatgtcccaagaattctctggaagccctatggcactgatgatcggaggcccaagagttggtagcagggttcttgagagaagtggaaataactcaaagccatatattcctgtgcctagatcccagggcttctttccaccaaggggttcacagtcacgaggtcctccatatatccctacgcttagatcagggataatgatggaagtgactccaggaaatgctagaatggcaaacagaggcaacatggctcatgtttctttcccactgggtagcccaaggcaccctatggataattggcaacagagtccttctctccctttgtcacccagtattacaggcttaccttgctcttctgctcattgctttctgcctccccaagctccagcattcaatccatttcctgtgatgcctactgcttttgcttctccactgcgatttggtcctccccttttgccttatgtttttcattacaacactggagcaatgtaccctcctccatacttaaactagagagtttgcaaagagaaaaagggttggggaaagagttgaacattactcactttcttcatatataaaattctctatccttttacattttgtttagcactaatgtgccttatgtatttagagatctaataaagattccacaattttaaatgttctagcttattttttaaaccaaaaccttaatgtttgttgtctgtgtttcaggaaattagtgtatatatatatatgtatatatacactagcatttcctacagagcttacttgaatcattatcattgtttgcccctgactggtgctgcaatgtgactctggaaaaatagcaatggggaccttgttagcaaagtctagagtctgtaatttctacatgcctaaagctaggatggttgcTCAAATTTGGATGAATGCTTTAAGGTAACTTTCACAGTGACAGGAAAGAGTCTGCAGAATCCGAAAAGATGCACAGAAAATAGAATGAGTCACCTGGGAAAATGTATCTTGATGATATCTAACTCAAATCCACCCTCTACTCTGCCTGGACAGAGAAAACATTTAATAATCAGATAAAATAGGGATAAATGATCATAAAATCAGGAAAAGTCATATTTCAGGGATT
This mutation is a 2206 bp deletion beginning at Chromosome X position 45,090,823 bp and ending after 45,093,028 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62687 | GTT CCC TGT TGT CTG CCT TC | Common | A | |||
| 62688 | CCA ACT GCA TTA TTC CAT ACT CAG A | Wild type Reverse | A | |||
| 62689 | TGC AGA CTC TTT CCT GTC ACT G | Mutant Reverse | A | |||
| 62690 | Fluorophore-1 | TGC CTA GCA ACT GAT GAC TTC ACT A | Quencher-1 | WT Probe | ||
| 62691 | Fluorophore-2 | TCA CTC CTT TGC TCA AAT TTG GAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62687 | 0.40 uM |
| 62688 | 0.40 uM |
| 62689 | 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.