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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
>chr4:108266719-108266835 117bp TAGGGAAGCACTGGGACGTA CCCAAAAGAGAACTTTCCAAGA
Mutant= 114 bp
Wild Type = 117 bp
Sequence
TTTGGTATCCAGATTGATAGAGACCAGACCTCATAATACCACACTTCTAGAGCTGAGAAGAGAGCATGGAGAATCACCATTGGCTCTCGGGTGCCTAATCCTACAGCGCCTTAGCGCTACCTGGCACGTGGTGCTGTCTGATAGTAGGGAAGCACTGGGACGTATCGCTGCCTAT^G^gtatctaatagtgaatctccgcaatgtctgttccattctgattctgctgttactagctccataatcttggaaagttctcttttgggtcttagtctagacaataatatcttcatctcaccaaattgtatcaagtaagtaaaatgcagctgcatggaattatgttagtgtggtgaaatttttgctcttctttgtctgtgattttgaagttaaaatgctgcttatttttccccaggtctcctgaatgatggaactgtgggaattttcagaggtaaccagatgcgcctaaagcgagcttgcattcgcaaagcaaagatttccgctgttgctttccggaaagccttttgccaccacaagctagtagaacttgatgccacgggtgtgaatgctgacatcactatcacagacatcataagtggccttggcagtaacaagtggatccagcaaaatcttcagtgcttagtactaaactcattaactctgtccctcgaggatccatatgagcggtgcttcagccggctctctggtcttcgagctctgagcatcactaatgtgctcttttacaatgaagatctggctgaagttgcttcgctgcctagactggagagcctggatatttctaacacctcaatcacagatatcactgccctgctggcctgcaaagacagactcaagtctctcaccatgcaccatctgaaatgtttaaaaatgactactacccagatcctggatgtcgttcgggagctaaaacatctgaaccatcttgacatctcagatgataaacagttcacgtcagacatagcccttcgattgttagagcaaaaggacatcttgcccaatcttgtgtccctggatgtttcagggagaaagcatgtgacagacaaagctgtcgaagcctttatacagcagcggcctagcatgcagttcgtagggttgctggctacagatgctggatactctgaattccttatgggcaaaggacacttgaaggttagatattaaaaattgtattttgcttgactggtcaggtttttttttcctagttccttaagacttcatTAGGGTAAATGTGTAATATTTGAAACACTCTGATACAAGGTATGGAAATAATTGTATAGTGGAGAAAGCCTTACATTAGGGTAATAATCCCAATTAAAAGTCCAGTGGGACCAAGCATGGCGAATGCTTTTAATCCAAGCACTCACAGAGCAGACGAAGGTAACTTCCTGCATGTACATAGCCTGGTGTACATGG
This is a 1055 bp deletion beginning at Chromosome 4 position 108,265,749 bp and ending after 108,266,803 bp (GRCm38/mm10). There is a single bp insertion (G) at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47463 | TAG GGA AGC ACT GGG ACG TA | Common | A | |||
| 47464 | CCC AAA AGA GAA CTT TCC AAG A | Wild type Reverse | A | |||
| 47465 | CCC TAA TGT AAG GCT TTC TCC A | Mutant Reverse | A | |||
| 47466 | Fluorophore-1 | AGT GAA TCT CCG CAA TGT CTG | Quencher-1 | WT Probe | ||
| 47467 | Fluorophore-2 | CTG CCT ATG GTA GGG TAA ATG TG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47463 | 0.40 uM |
| 47464 | 0.40 uM |
| 47465 | 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.