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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
>chr4:88721927-88722011 85bp GCTTCTCCCATGAAGGTGTC ACCGCGTAGTTCCAGACTTG
Mut = 104 bp
Wt = 85 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TGCGGCCTGCAGCTCGCAGCGAGCTCCGGCTTCTCCCATGAAGGTGTCTCTGGGtaacggcgacatgggcgtctctgcccacctgcagccttgcaagtctggaactacgcggttttttaccagcaacacgcacagttcggttgtattgcaaggctttgatcagctcagaatagagggcctactttgtgacgtgacgctggtacctggcgatggagaggaaatcttcccggttcatagagccatgatggcgtctgccagtgattatttcaaggccatgttcactggaggaatgaaagagaaagatttgatgtgcatcaagcttcatggggtgaacaaagttggtctgaagaaaatcattgattttatttatactgcaaaactgtctcttaatatggacaatcttcaggacactcttgaagctgccagctttctacagatcttgccggttttggatttttgtaaagtatttcttatatcaggcgtctctttggataactgtgtggaggttggccggattgctaacacctacaatcttatagaagtggataaatacgtaaataatttcatcctgaagaactttcctgccttgttgaatactggggagtttctgaaactcccctttgaacggcttgcctttgtgctttccagcaatagtctcaagcactgttcggaacttgagctcttcaaggcagcctgccgctggctgaggctggaagaccctcggatggactatgccgcaaaattaatgaagaatattcgctttccactgatgacaccacaggatctcatcaactatgtgcagacagtagatttcatgagaacagacaatacctgtgtgaatttacttttggaagcaagcaattaccaaatgatgccgtatatgcagccggtgatgcagtcggacagaactgccattcgatctgattctactcatttggttaccttaggaggagttttgagacaacagctagttgtcagtaaagagttacgaatgtacgacgaaagggcgcaggagtggaagtcgttagcgcccatggatgctcctcgataccagcatggcattgctgttattggaaatttcctttatgtagttggtggccagagtaattatgatacaaaaggaaaaacggctgttgatacagttttcagatttgatcctcgttataataagtggatgcaggtggcatcattaaacgaaaagcgcacattctttcacctgagtgccctcaaaggacatttatatgcagttggtgggcgcagtgcagctggtgaactggccacagtagaatgttacaacccaagaatgaatgaatggagctacgttgcaaagatgagtgaaccccactatggccatgctggaacagtatatgggggtttaatgtatatttccggaggaatcactcatgacactttccaaaatgagctcatgtgttttgacccagatacagacaaatggacacagaaggctcctatgactacagtccgaggtctgcattgcatgtgtacagttggagacaagctgtatgtcatcggtggcaaccacttcagaggaacaagtgattatgatgatgtcctaagctgcgaatactattcgccaactcttgaccagtggacaccaattgctgctatgctgagaggtcagagtgatgttggagttgcagtctttgaaaacaaaatctatgttgttggtggatattcttggaataaccggtgcatggtagaaattgtccagaaatatgacccagaaaaagacgagtggcataaagtttttgatcttcctgagtcgctgggtggcattcgggcttgcactctcacagtttttccacctgaagaaaaccctgggtcaccttctagagaatcacctctttcagcaccttcagATCATTCTTAGATCCAAGGTGTAATACCTTTTCAGTGTATCATGGGTGGTCTTGTTCATCCCCTTGTATTGTTTCTCCTTATTGCTACAGTTAATAGACAAAAAGGTAACTTAGACCCT
This mutation is a 1826 bp deletion beginning at Chromosome 4 position 88,720,160 bp and ending after 88,721,985 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51077 | GCT TCT CCC ATG AAG GTG TC | Common | A | |||
| 51078 | ACC GCG TAG TTC CAG ACT TG | Wild type Reverse | A | |||
| 51080 | Fluorophore-1 | ATG GGC GTC TCT GCC C | Quencher-1 | WT Probe | ||
| 52620 | GGA GAA ACA ATA CAA GGG GAT G | Mutant Reverse | A | |||
| 52621 | Fluorophore-2 | CCT TTT CAG TGT ATC ATG GGT GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51077 | 0.40 uM |
| 51078 | 0.40 uM |
| 52620 | 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.