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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= 133 bp
Wild Type = 151 bp
chr19:5364436-5364586 151bp GGGCTGAACACGTCTACTTCA CCACACTCTCTCCCACAATG
Sequence
Wt Sequence (deletions in lower case): GAGGGGCGATGCGCGGTGTGTCCGGGTGGCGGCGCGTGGAGGGGGCGGGGCGCTCGGAGCGGGGCGGGGCCTCAGGGCGGGGCCTGCGGGACCTGAGAGGCGTGGAAGAGGCCGGCAGCCCGAGTGAGGGCGGCGAGGCTGGCGAGAGCCGCGTGCTGTTCCTACCACGTGTTAGTTCCCAACGGGGTCAGTGTCTGTGTAGCCAGGGTCGCGGGCTGTGAGGGCTTCAGGGCGGACCTTGACTACTGAGTCTGGGAGTCGCTCTGTTGCCTTAGGTGCGCCACCAGCTAGTGCAAGCTGATATATCCAATATACCCAAAACCAGGACCCACGGAAAGTGGAAATTTCCGAATAgccggggatgggttggggggactcaagggagcagccacagtagctgtggtaggatattgcccttgggacttgtggtcttcagccctaacctacttttcttttttttttttttttttttttggcgggggggattcccaggttaagcctgatcacgatgacaacctcccaaaagcaccgagacttcgtggcagagcccatgggggaaaagccagtggggagcctggccgggattggtgacgtcctgagcaagaggctggaggaaaggggctttgacaaggtgtgggggggctgcaaatgccagggaggggtaggagcgaaggttggcaacaggtggtaccacgcagattgggagtgaggcccacacatgtttggggggtacctggggaggcatggtgcctccttcatttttgtgttttgttgagacagggcttctcagtgctaggattaaaggtgtggctcactgtcccctgcccttttgtgtgttttaaatatcacgaaaatgaaactaggcatagtggattgtgcttatgaccctaacacttgggaagttgagctaagaggattcccgtgggatttaatgccagccctgacaacaaaggaacaaaccctgtctttctttttttttttttttcgagacagggtttctctgtgtagccctggctgtcctggaactcactctgtagaccaggctggcctcgaactcagaaatccgcctgtctctgcctcccaagtgctgggattaaagtcgtgcgccaccactgcctggcaacaaaccctgtcttaatcaaaataaaaatggccttgccctctgaagtatttaccacctaaccctccctagcttcagagatctggcctcttcggagtggtatcagcgtggacagtcctgttgctttagcccatgagacacttagccacatgcccttttctattctgcaggcttatgtggtccttggccagtttctggtgctaaagaaagatgaagacctcttccgagaatggctgaaggatacatgtggtgccaatgccaagcagtcccgggactgctttgggtgccttcgagaatggtgtgatgccttcttgtagtgttctctggggatccctcataccccagccacagcacagtttccagagtctgcagcagggcagggctcctccctgtcctccataaaggaaaagatggctgttgtcaccctcacctcctatgtactccagggtctatggggagttctctcccctcagcgtttcaatctttctgggatcgtttcccttgcatgcctttcccttccacctccccctgcagttccgtggcagcagttaccaactttcctgaatggattcctggcccccttctccctcactatctgttggatgccctttggctcctttcctttttttggcaaaaacagtcactgtccttataaaagatttttaaattaataaaatcagtggccttcatacttggtgttctgtgtcagagagcagataggctggggagttgtgtattcccagggctgaacacgtctacttcattcgttatcagtgcagaccgaaactggtgccttctatttcccctcccaagagggagccaccctagccatgtgggctctgggcaggtgccctgGGTGTCCCAATTCCTAAACATTGTGGGAGAGAGTGTGGGTTGGCTTTTTTCTCCTGGGAGCCAGGTAGGCTGAAGGCTTCCAGATGTCAGCAGTCAGTCTAGGCAGCACCTGGTGGAAAGTCAGTGGGAGTCAAAGGTACCAGGAAGCGGGTGGATGGGATGTGGGTAAGAGGTCACCTGCCAGTCATCATAGCATGTGCTCTTAGCGTCTGGCAAGAATGAATGGGTGGGACTTTTGCTGTTTGCCAAGCTACCATAGAAAAGGAAGGAAATGGTTAAACTTGAAAGCCAAATCCTACTTCTATTGTTTTCAAGCTCATATTGTGAGCCAACCCTGAAACTAGTATCTTTGTAAGCACTTATTACATTTTTTATTTTGGAATGGTAAGAGATTTGGAGCTTAGAAAGCTAATTTGCCCTTGAAC
This is a a 1621 bp deletion beginning at Chromosome 19 position 5,414,502 bp and ending after 5,416,122 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59518 | GGG CTG AAC ACG TCT ACT TCA | WT Probe | A | |||
| 59519 | Fluorophore-1 | AGC CAC CCT AGC CAT GTG G | Quencher-1 | WT Probe | ||
| 59520 | CCA CAC TCT CTC CCA CAA TG | Common | A | |||
| 59521 | CGC TCT GTT GCC TTA GGT G | Mutant Forward | A | |||
| 59522 | Fluorophore-2 | ATT TCC GAA TAG GTG TCC CAA TTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59518 | 0.40 uM |
| 59520 | 0.40 uM |
| 59521 | 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.