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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
>chr1:163997479+163997574 96bp ACATGTTCCGAGGGAATACT ATCTAGCCAGGCACTTCAGG
Mut= 97 bp
Wt= 96 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (GTAGTATGTAGAGTA insertion):
CTTTAATCCCAGCACTCGGGAGACAGAGGCAGGTGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGAACAGCCAGGGCTATAGAGAAACCCTGTCTCGAAAAACCAAAAAAAAAAAAAAAAAAAAAAAGACCAGGCAGCAGGTAGCTAGGCTGTCCTGTCTACA^aatcttggggtttattgttctggtgaaggtggtagctgctatagctattcttttgtttgtctttatgttttatcttctacttttgaatacttaaggcatctgtgcattttgctaaatcgcatacacacagcctttttttgtttttgtttttaaacattttcttttcccccttccctttccagggctgaactgaaaaaggatcaaaggcgtctaaaacgtctttggagccccctctgaatacatctgatggtgtatacgcagctaacagacattgggcgcaacagaactaatcgactgtgtgatcggttatgccattaaattctttctagaagctgaagcatcatcctttggtaaaaaaacggtgtgcatagaaaaagaggatggcctttcaattcaatttcagcatagaagaagatctggaaaataaattaacatccctcgatgatggaacttgtgtcttagaaagtcagaaaggaaagcaggacaaaaggcaatctacagaacgtccgggcttgcctcgggatcactcttggaaatgctcctcactgggaaatgcagcttcctctgaagacactggcagcccaccccctattgctgacaggtcagatgacccagaagcttgtaaacaccagccctcctggaaacctgctaaagaacatgtgatgcctaaagatgtgaatcacgtcttagaaaatacggttttggaaatgttgccaggtccccagcatgctaacacagcagtagtgaaaaccgtctctttgaaagagaaattccctggagaaaacatcgtttcaaagagcttttcttctcattctgatctgatcccaggtgtttatgaaggaggcttgaaaatctgggaatgcacctttgacctcttgacttacttcacaaaagccaaagtgaaatttgctgggcagaaagtgttagaccttggctgtgggtctgggctgcttgggataactgcatccaaaggaggggctagggaagttcattttcaagattacaacggtttggtgatcgatgaggtgaccttacctaatgtagttgctaatgtccctttgcaagatgacagcaatggcaaaaatgagccagatgggaaaagacaaaggaagtcagaagtaggccgagaaatctgtaaatgtcgcttgttctctggggagtgggctgagttttgcaagcttgtattacgtgaaaaactgtttgtgaaatatgacctcattctcacttcagaaaccatttataatccggattattacagcactttgcatgaaacattcctcaggctattaagtaggagtggccgggtgcttctggccagcaaagcacattattttggtgttggtggtggcgttcatctcttccagaagtttgtagaagaaaagggtgtatttgagactcgaacacttgaagtaattgatgaaggtctaaagagatttctaatggagatgacttttaagcaccccagttaatatgcaccgaatggcgtaatgaaataaaaagaataccccagaatttgtgactgttcaatttcttttttattgttattctgaaattacggtttggtgttttttttttttaatttacttattgaaataataagcaaagtatagaagtagtagcctcatgttttctatgtaaaactacaagtttccttgagaatttaatgagctcttgaatcactttgtgtaaaagttctttctccatcctcattctatttgatctttctacaatctctaacactccataaacatgttccgagggaatactcccatttcttgacaagtttatg^AATCTCTCTGCAGCCTTTGTTAGTTCCTTGTCTCCCTGAAGTGCCTGGCTAGATCGATACAGACTTTGATACGGACTTGGATCCCTGAATAATACTGACCTCAAGCGTCTGGTCCAAGGCCTGTGTTAACTCAGAAGATGTTTATTGATTTGCGTATGTCAAAGTGTTAGCATACTAGGCAGTTGGAT
This mutation is a 1789 bp deletion beginning at Chromosome 1 position 163,995,732 bp and ending after 163,997,520 bp (GRCm38/mm10). In addition, there is a 14 bp insertion at the deletion site (GTAGTATGTAGAGTA).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47635 | ACA TGT TCC GAG GGA ATA CT | Wild type Forward | A | |||
| 47636 | ATC TAG CCA GGC ACT TCA GG | Common | A | |||
| 47637 | AGC AGG TAG CTA GGC TGT CC | Mutant Forward | A | |||
| 47638 | Fluorophore-1 | CCC ATT TCT TGA CAA GTT TAT G | Quencher-1 | WT Probe | ||
| 47639 | Fluorophore-2 | CTA CAT ACT ACA ATC TCT CTG CAG C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47635 | 0.40 uM |
| 47636 | 0.40 uM |
| 47637 | 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.