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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
>chr2:119210298+119210382 85bp ACTCCGGAAACAAGGAATTG GTGACCAGATTTTGAAGGAAGG
Mut= 83 bp
Wt= 85 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGAGGCGAACCTCCCACCATCTCTACCAGAACAGAGAAAGCACGGGATCTGCACCCAACTCTGATTCTTTTTGTTGACAAGCCAGGGACCTGACACACTGGGCCTTGTTTGTCTTTGCACTACACCCCCATGGTAAGAAATTGAAAATGGTAGAAAGCATGTGAATTTTGCCTGAAGTTTTATCTAATTTGAATAAGACTCCGGAAACAAGGAATTGCTGAACCCATCTCGGCCAGAAccctagggataccactcttcctccttccttcaaaatctggtcacaacttcacttccttctttctgtgagtgcttagagggtctcagagaagagccatcggggctgggtgagacatgcttcctcctgcttcctgtttcttatagtatggctgtaagaattgtggccgagccttctgtaacggctgtcttagcttcagcgctctggtaccccgggctggcaacacacaacagaaagtctgcaagcagtgccacacaatcctgaccaggtgagagatgggctctgacaagggttcagacagacatgacaataagcatctggtcaggccttgtggccttgtgcgccctggccagaggtctgcctgggaagtatttggggtagaaattaccaatgggaaggagggcaggacttctgacaccattgtcctctgatgtcctttctccagagggtcttctgacaatgcttccaagtggtcaccacctcagaactataagaagtaagtgctgagaagaagacaagtggggctgggaagaaacaaggacaccactaccacatctctccttccagtctccagagcaaatgggtgcagggcagagagaatgcccacctccaggctcctggcttccctcttggatcctgaaagctgctcattaaccagttatcagaaaaggcatttgggagctccttccctgtcaggcataccctcctgctggtcatctgggttaggctgtggtaccacatgaatagaggccacagaagatggcctgtagaggcccagattgatttgaatgggtgccatcagtcttctctttaaccttaagggtctctacctcatattcacccaggcgtgtggcagccttggaagccaagaaaaagtccagcacttcccatagccaaagcctgacccacaaagaccaagccatcgctgagcgcctagcacggcttcgtcaggaaaacaagcctagtgagtgggagtgggccaggggacctttaaaggcacataccagaagtgaggtctgagcttcctctggcccttacaaatcttctcccttccaactgcagagtcagtaccctcacaagccgagatagaggcacggctagccgcactgaaggatgaagtccagggccccattccttccacgcaggagatggaggatcggcttgcagccttgcagggcagagtcccaccttctcacaccgtgagaccggtgagtgtgtggcttagaagaaaaggatttcctagggtgagcacccttcacaagcccagatacatgtatgatcctgtgctttgcagggattggtaaagcctcccacccctacctcctgagccttaagcctgctcctaagccaaagaacaaaggccaagcagcaggactgagtgcgtcagcagagtggatactcagtcctgttgggagccgccagaccgactcacctatgtggacttgaattcatccctttccttgggctttgaaaattcttcagcatctacccctgactcagacttcttctgcactcttggtgtggccttccccacagccctttccccactgagctgccctcctccgcacaggcacaccaggcaccagataccaggacccaggcccagcagacgcaggatctgctaacacagttgacagctgaggtggctattgatgaaaactgccaaccaagagcctcaggtaacttgtccccttggcttctcccaagtcatgtcctgcctctgatacccacccttttgggagGTGAATGTAAGTCAGAGAATAGTCAGCACAAAAGACCTTCTGTTTACTGAGGGCTAGCTCCAAAGAGCCAGCGTGGGCCTCTTGCACTGGCTCTAGAATCAGATGGTGAATTCCACTTCCACCACATACATGAGTTACCTCACCTGAGTCTTTCTCATCAGTAAGCTTAAGCTACTGTGCTAAAACCTATGTCCTGGGAGAAATCATGAGGACTAAATGAAACAATACAGTAAGGGCT
This mutation is a 1739 bp deletion beginning at Chromosome 2 position 119,210,339 bp and ending after 119,212,077 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52743 | ACT CCG GAA ACA AGG AAT TG | Common | A | |||
| 52744 | GTG ACC AGA TTT TGA AGG AAG G | Wild type Reverse | A | |||
| 52745 | CAG AAG GTC TTT TGT GCT GAC | Mutant Reverse | A | |||
| 52746 | Fluorophore-1 | ACC CTA GGG ATA CCA CTC TTC CT | Quencher-1 | WT Probe | ||
| 52747 | Fluorophore-2 | CGG CCA GAA GTG AAT GTA AGT C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52743 | 0.40 uM |
| 52744 | 0.40 uM |
| 52745 | 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.