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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
Mut= 92 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
X-Linked
>chrX:153037744-153037839 96bp CCTTTGGTGAGACTGGAAGG GCAAGAGAATGTCGGTCTTC
Sequence
Wt Sequence (deletions in lower case):
CTTCTGAAAACCCAGGCGGGAGAGTGAATATGCTGTGTAAGCCAAGGCAGCCAAGGAGGGCTGGGGTCAGCTGACCTCTTGCAAGCCTGTGCCCCGCCGCACCCCTCCCCCACCCCACCCCACCCCTCTCAGCCCGCCTTTGGTGAGACTGGAAGGGCCCACTGCTTGCCAAAAGCTATgaaccaataggataatgtgtgcacctgcctaaagaagaccgacattctcttgcgcgcaggacaaatccctgcaaccccccgccccctagctgtcttaactaccattgtgggactcaaaatgcagcccgctccttttcctttcggctagcggaagttcctgtgcgggagagtcccacttcctcttgggtgtttggcgtgtgcgtggcaggaggggcggggccaatgctgctctggtttcttgcgcgcttggcgctacagggagtgcgggcggcgactccttgcgcaagtcagcttgcctgggaaagggctttgtggctgaaagcgactggttccttgccacaaaggctccgctggcgtttgcggttcagcggccgtccctgagtaagatagccacttttctccgacgctgccaatagccttctccaagtgctgcaggctttcatcgctttgcaggagccatgcctcggggacggaagagtcggcgccgccggaacgcaaaggcagctgaagagaatcgcaacaatcgcaagagccaggcctcagaggcttcagagaccccgatggcggcttctgtagccccgagcacacccgaagaatacctgagcggcccggaggaagacacaagcaccctggagaaggcctccagtaccccttcagaagcttcgagcactgccctagtgcaaaagccggttacccggagcaattttcagggcaccaagaaaagtctcctaatgtccatattagccctcatcttcatcatgggcaacagcgcaaaggaggccctggtgtggaaagtgctggggaagttagggatgcagcctgggaggcagcacagcatctttggagatccgaagaaggtcgttacagaagagtttgtgcgcagagggtatctgatttataagccagtgccccgcagcagtccagtggagtatgagttcttctggggccctcgagcacacgtggaatctagcaagctgaaagtcatgcattttgtggcaagagttcggaaccgatgctccaaagactggccatgtaactatgactgggattcagatgatgatgcagaggttgaggctattctcaattcgggtgctaggggttattctgctccatagagagatcctaggcaaacccttaggagtggaaaagagaatccaaagtatctgctccatggagtagataggcagggtcctgaattcaaaaggttgtggagggtggggagggcactgtatttggtatttgtgaccagtgctaattgttaaactgcaaagtagtgtttgctatagatattgttatcttgtattcattttataacactgttgactaagaatcacaatatgtttaaatatatacttgaggaaaagtttatgttatttttttctgtcgttggcatttgatattaacagctgtgctaaccttattaaatgaagtctttgtgtcatgttgtgtgatggatagaaagtaccactgagaggctgttctttgaaagtttggaataaccagtaaagtgtgagtggaagaagattacagatttgttctgtctttcccttgtgtctgatgtcttgtaatggaacacatttgtaaatatgctcactgtgcaaatagtataaataaaagcatagtaaacagaatgttttgttcatttattgtccacctgttaaatgaggaccaaatttagatgctgggaatagtccttaacagaaagcatctagtttccaagaaatttcaagattgtgggagggggtggggaatggaaatgcagtaagtgttgtagggaatgggcaggGACTAAGGAGAAGGAAGACAAACTGATGACATCATTTGGGGAATAGAACAGAAAAAAAGAGGGTATCACAGGATGCTTTAAAACCCAAGGAGCATTTCTAGAATGCAGAAGACAGTAGGAAGGGTGGCCATATTACTTGGTATTGAGTGTGGAACTGGATACTTAACAGACCTTGACAGGAGCCATTGGCAAAGACCTGAAAACATTCTAAAATAATAAAGAAGTAGAAACGACACTAAAATTATCTTGAGAGAGGATGGTAGGGTAAAAATAGA
This is a 1786 bp deletion beginning at Chromosome X position 153,036,011 bp and ending after 153,037,796 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 60758 | CCT TTG GTG AGA CTG GAA GG | Common | A | |||
| 60759 | GCA AGA GAA TGT CGG TCT TC | Wild type Reverse | A | |||
| 60760 | GTT CTA TTC CCC AAA TGA TGT C | Mutant Reverse | A | |||
| 60761 | Fluorophore-1 | CCA ATA GGA TAA TGT GTG CAC CT | Quencher-1 | WT Probe | ||
| 60762 | Fluorophore-2 | CCA AAA GCT ATG ACT AAG GAG AAG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 60758 | 0.40 uM |
| 60759 | 0.40 uM |
| 60760 | 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.