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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= 120 bp
Wild Type = 114 bp
chr2:157556069-157556182 114bp CAGGCATTAACAGGAGGGAGT GGGATCCACTCCTCAAGTCC
The Wt For primer (primer 59969) anneals over the nucleotide sequence containing mouse genomic variations rs27338091.
Sequence
Wt Sequence (deletions in lower case): GGCTGCAGCCCCAGGAGGCGATACATGATCTCTGCTGGGCAGGGCCCAGGCAAAGCCAGCCCACTGTGGGGACCGTGTCCTCCTGGGATCCCCCAGTAGAAGCctagggaagacacaccctagcaggctatctttaagtaataatttggataggagtagacattcatgggtgggtggggaggaaagggcattcacatttgctgttgttaaccatgatccattttcccaaataagtatacaatttgttcctgtttttttgtttgttttgtttgttgttttttgttttttgtgtgtgtctgtctgtcctttgtctgtttgttgtgttgacttttctcttcaggatccctgctgccttggtgatcccgggctgacagccagagagcacagcggctcagctcctggagagagagtcgaagaaagcgaagggcagccacctgtgcctgctggctcccattaggtcggttcctgcagcggtgcctggcagccttggtgaaggccctgcccggcagagatcatgtattgcctccagtggctgctgcccgtcctcctcatccccaagcccctcaaccccgctctgtggttcagccactccatgttcatgggcttctacctgctcagcttcctcctggaacggaaaccttgcacaatatgtgccttggttttcctggcagccctctttctcatctgctatagctgctggggaaattgtttcctgtaccactgctccgattccccacttccggaatcagcccatgaccccggtgttgtgggcacctaacgtctgccgagatagcttgccaaggaagcagaagacgggaggggaggcattgacataggtcataaagcattggagtttcaaatcccgcagtcctgcgggtaccacattcctaatggagccttttggcctgtgatgttttatccttacaatgtgaataatggcactgatcggtgcttctgttgtagagtcctgtagtcgtgggtggtcttatggttgtgtgttctgtcaccatctgggtcccggctgacggattggcccacccccttgctcattgatttggggaatctatacccttgatatgacctgtgtggatacagtgtagtctcaatgtcacctccataacccttcctcgtcaagaccttcctcgttccctcctcgtcccctttccccgttttccccttggttcacttccaacccctttccttttttggggagcacctgtccaagacagggcttgtttttgcacttatctcaaatttgaagagattgctgatgcccgagagcctcgctttttcatccttcgttcccttttgagaaggtgagacggaatcatgtctcaactgctcgttgtctgcagacctccagtatttcctctgcctcatttttaagaaagaagcgatggggagacattgctctttggcctgggtatctgggctcctgccttccagcccagcctctctccctttgctcttcctcctgtctctctcagctgacctaaaggggccacctcatgtctccagtgcatgctcttcaggagggagatgtgcagtattctcgtagacccagtggtccctggctgagtgaatgagaaagtattacatttttcatagcagccatgattcccttggtaggtgtttggatatttttgatgtgccctgtatgtatgtgtgcactagtgtcagcgtgtatgtacacacgtatgtgtgaatgtgtgttgtgtgtgtgtttacataccaatacatgtgtatattccttttgaagaagctttattgaatatgttctgattttgaggtttagtagtagtagctagctgtagtaggtcctgctgcagtttttatttagcatggggattgcagagcgaccagcacagtggactccaaggtggttcagacaagacccaggggagcagtcgccatcatcctcccaccaggagcttcctcattgctgcgcacgtagactgtacactatgaagaaaacacaggaagaaagatttggtgacttggtacttgtttgcttttctctgcgcttcagaaacaagtgtttgcaaatgagactttctcctggcccctacccactggggatcagcatggttgttcttccagtcggaaatgtacccccctcctttccccctcttgtgtgcaagtggggggggggggcaggcataggacagagctggaagcaggcttctggggagtgggacttagaggccacacttgtgaaacactcggactgctgttgtaaagcttttatttctggtgtgttcgttccacagctgtttgaaatgtttaataaagctttataaactttactttgtggttttatgtggctgcagtccatttgggtcatggtgttggatttctgagttggtggggggcagggtgaagcacacagggctgagtccttggcgccagagtaccttagctggaaaatctgatctgttctctggagaacagatgctggaattggggaggaaagcagggggcagctcaggcattaacaggagggagtctggaagttttcagagatgccatttgcacttctcagctcccatggcttcctgcctgaagcTGTCTGCTCTATAGGACTTGAGGAGTGGATCCCTCGATAAACAACTTGGGACAGCTGGGG
This is a 2463 bp deletion beginning at Chromosome 2 position 157,398,022 bp and ending after 157,400,484 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 59969 | CAG GCA TTA ACA GGA GGG AGT | Wild type Forward | A | |||
| 59970 | GGG ATC CAC TCC TCA AGT CC | Common | A | |||
| 59971 | GGC GAT ACA TGA TCT CTG CTG | Mutant Forward | A | |||
| 59972 | Fluorophore-1 | ATC CCC CAG TAG AAG CTG TCT G | Quencher-1 | MUT Probe | ||
| 59973 | Fluorophore-2 | AGC TCC CAT GGC TTC CTG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 59969 | 0.40 uM |
| 59970 | 0.40 uM |
| 59971 | 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.