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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
>chr4:141118260-141118368 109bp GGCTGCAAGGTCAAAGGA CAAGGCAGAATGACCCCTAC
Mut= 92 bp
Wt= 109 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CACCGACAGCTCAGTCCTGTTCCAGTGCAAAACAGGAAAGGTAGATTCAAGGGATGAGGGACGCAGAGCTCAGATGTCCTGAGAGCGCTTGAGTGGGAAGATGGCTTGGTTAGCTATGTGTTGGGAAAGTTGGAGTGAGTCTGAAGGAGACAACAGGAGACTGGCTGGAGACTGTGCAAAACAAACAAACAAAAAACACCAAAAACCCCCACctaatgatggggtccagaactattgctgtgctatgctcatggggctaaggggagatggaccttcagaggaagccaggtccccaaacctcaggtccaggtgaaggacacagccaagtccctgcctagagagaagacactgtccttagcttggaaaaaaataaagacttaaggttttgagagagattttttttccacttttatatacatttttttcctctaggaaatagacagacggttggaaaaaaaactgaagatcacacaaaaagagaggtaaggctgctgtgtggcgtgagggctcccgctctctgccgctggagatctgggcaggcagtattctccggctggctgctagtgttaggtggttaggctcctgggcgtcagcttttccctcccagtaggcgatgaggatgctggattgaggctttggggtgaaatgtattgcagctgtctccagcctcagtggaccctgtgtgagctcgtctgatggagggaggtgacaggatttgggtttatgtgaggctgtgcccagtggctccgaagtgtggagtagttgtttctccctctgggtattggtgtcttttgtaacacagagagtttggacagataattcctctgtgtccttgatggcttgacagcttagagtattgtgaggagatctcagaaggaaagattctagaaatcctcgggaaacctttaaggattgttttctcgccattgtgtagcgtggttagtggggtccagagtgagtgactttgagcttagtaacccagggacaagcaacagaaagaactgatttaccccccagggcattctgggagtccacactgactttccagactccctgctgctgggatcagactgctggctctctctgattcccaggagctacttcccatggttccaggcccctttccatgccgccaccacccacctaggacccttgcatctgacacagagcatgtagcttccaccctcctgacagttgaggccctggcaatggtctcggtggttgtgaacaagagctggggcggcaccggtggctggtgtaggtgttctccttgccttgctcccagtctcatctgtggcttctgtttgctccctccctgtgctgttggtcctctttgagttggggaatgtcacctctgcttgtggccacctggcattgatgtgcatatctaccagccttggacaatgcttctcttttgagcattgccaagtagtttcatcccagccccgccgctgcccaatagagcagagagccctggtaagggagagttctctgctcagaggctcttgtgtggaggtcacttgccagaaggcagagagagcttggtaaatcttgtcttgcttgttgccaggttgggcagcacagtaggtgcctgtgtactgtctcagttttgagcatacgctgtgggagacctgggctgagggacaccccagttcaccagcctcctacactctggtctgtgtagggaagctggggttttcatccaagtcgttgaattttgggtgtgaaatgttttggcttcgagggcaaaatgatgcttgctgggcctgttaatgcacatggagaggaagtcccaggaccacaaggtggtgacctgcagggttagatctctggaacagctctctgccctgcagagctggtggcagggctgctgtcctgcctgaggtgtgggagtatagttgtctggtcctgggccaggtgactagttagcaggaagggagaagacagtgaggaggagggggtggggctcctatgggccttctgcttactgtgggattcatgaagctatgtttgctctttgtcagcaggaaatccaaatctcctcccaaagtgcccattgtgattcaagacgatagccttcccacggggccccctccacagatccgcatcctcaagaggcccaccagcaacggtgtggtcagcagccccaactccaccagcaggccagcccttcctgtcaagtccctagcacagcgggaggcagagtatgcagaggctcggagacggatcctaggcagtgccagccctgaagaggagcaggagaaacccatcctggacaggtgagtgtagtgcgggcagtggcaggggaggaaccggcctgctgggcaccgaactgaggtccttagctcagagccaaagcactatagcagtgtagttctctccggcccagggcgctgtcaggtgttaagtgtggtggctaatgggtccacagagtgcaggctgcaaggtcaaaggatgggcttcgagtcaggctctgtcagctgcacgctacgatcacttaaccTGATGGCTGATCTCAGAAGTAGGGTAGGGGTCATTCTGCCTTGCCCACCAGCTTAGCAGGGAGCAAAAGGCCATAAGGTGCTGAATTCTGCCCAGTCCCCCAGAAAAGCCCAGTATTGCTGGCTGGGTCTCCAGCAGCCCTGTGCAAGTTTTGCAATAATTTTCACTTGTGTCCCTTTTCAATTTTTTTTCTTTCTTTCTTCTTTTTTTTTTTTAAGATTTTAG
This mutation is a 2291 bp deletion beginning at Chromosome 4 position 141,118,303 bp and ending after 141,120,593 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48994 | GGC TGC AAG GTC AAA GGA | Wild type Forward | A | |||
| 48995 | CAA GGC AGA ATG ACC CCT AC | Common | A | |||
| 48996 | GCT GGA GAC TGT GCA AAA CA | Mutant Forward | A | |||
| 48997 | Fluorophore-1 | CGA GTC AGG CTC TGT CAG CT | Quencher-1 | WT Probe | ||
| 48998 | Fluorophore-2 | AAA ACC CCC ACT GAT GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48994 | 0.40 uM |
| 48995 | 0.40 uM |
| 48996 | 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.