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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
>chr18:37303251+37303341 91bp CTGACAGGAGATGCTGGGACT TCCCTTAACTTCTTTCAGATTCTTCC
Mut = 115 bp
Wt = 91 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AAACCACTGCTTGTGTGAACCTGGGTAGATTTTTAAGCACAAAAATCTGAATCTCTGGAAAACATTAGTTGTTAGGCTGTCTCCAAAAGACCTTGCTTTTCACAGAGAGATATAAGGCCCCTCTTGGTGACAAGCCCCTGGAAATACACCGCAGAAATACAATGGAGGCCAGAGAGGAAAGCTTTCTTAAACAAAGGCAAGTCTTGCTTCTCTTTGTTTTTCTGGGTGGGTCTcttgctgggtccatgtctaggcgctattctgtggcggaggaaaaggagaagggcttcgtaatagccaatttagcgacggatctggggcttagtatagaagaactggctgaaaggagagctcaagctatctccaaagggaacatacagtattttcagctcagtcatcagaccggagatttgctcttagttgagaaattggaccgggaggatttgtgtggctcgacagagccttgtgtgctgcactttcagatattgctgcatgatcctttgcagtttattacaaatgagctcgaggtcatagatgtaaatgaccatgccccggagttctttgaaaatgcaatgcagctgaaagtcctggaaaactccgtgcctgggacagtaattcctttgggaaatgccgtggacctggatgtgggaagaaacggactccagaactacacggtctctcccacgtctcattttcatgttcacacccgccgtcgcagggatggaagaaagtatccagagctagttctagacagagctctggatcgggaagagcagtcagagatcagtttaactctcacagccctggatggaggatccccgcctcgttctggaactacccaggtccacatcctggtcttagacacaaatgataatgccccagaatttacacagtcactctatgaggttcaaattttagagaaaagccctattggctctgtcattaccactgtctctgcctctgacttggatacgggaaattttggtgcaatatcatatgtcttttttcatgcttctgaagaaattctcgaaacttttcgactgaactccagtactggtaatatacagctacttaagggtttggattatgaagcaattaatacttatgaggttgacatagaggccaaggatggtggaggcctttcagggaaatgcacagtcatagttcagatacttgatgtgaacgataacccaccagaactgaccctgtcatcagttaacagtcttatccccgagaattcagcagaaactgtggtggctgttttcagtgtttctgatttagactctggagataacggaagagtgacatgttccatccagaacgacctccccttcatcttgaaaccatctgtagagaacttctatactatagcgtccgaatgggctttggacagagagatcagagctgagtacaacatcaccatcacggtctcagacctgggcacacccaggctcacaacccagcacaccataacagtgcaagtgtctgacctcaacgacaacgcccccgccttcacccaaacttcctacaccatgtcggtccgtgagaacaacagccctgccctgcacataggcaccatcagtgccacagactcagactcaggctccaatgcccacatcacctactcgctgctgccgccccacgacccacagctggccctcgattcactcatctccatcaatgctgacaacgggcagctgtttgcactcagggcgctggactatgaggccctgcaggccttcgagttccatgtgggcgccacagatggaggctcacccgcgctcagcagccaggctctggtgcgcgtggtggtgctggacgacaatgacaatgcgcccttcgtgctctacccactacagaacgcttctgcgccctgcactgagctgctgcccagggctgcggagactggatacctggtcaccaaggtagtggcagtggaccgcgactctggacagaatgcctggctgtcattccagctgctcaagtccacagagcccgggttgttcagtgtgtgggctcacaatggcgaggtgcgcaccgccaggctgctgagtgagcgcgatatgctcaagcaaaagctgctgctactggtcaaggacaatggggatcctcctcgctctgcaagtgtcaccctgaatgtgctgctagtggatggcttttctcaaccctacctgcctttgcctgaggtggcacgcgactccgtccaaaacgacgtggacttgctcacgttgtacttagtcatcgccttggcatctgtgtcttctctcttcctcttgtctgtgctgctgtttgtgggggtgagactgtgcaggagggccagagcagcttctatgggtggctgctctgtgcctgagggacactttcctggccacctggtggacgtcagcggtatggggaccctgtcacagagctaccagtatgaggtgtgtctgacaggagatgctgggactggtgagttcaaactcctgaatccggtgataccaaacctgtttCTGGAAGAATCTGAAAGAAGTTAAGGGAAGTTCCCACTGTAGGAGCAGCTTTGTAGTCAGCTAAGAATTGTAAGTGGTTTGTCCTCTCTTGGTCCTGGTAAACTTCTTATTCTAATTATTCTTTTAAAAAGTTGTACTACCCATTTTTTTTCTTTATCAGTAATTCAAGCCATGGTATCCCAAATAGCTTTAAACTTATTATGTTCTTATTAATACTGATTTT
This mutation is a 2259 bp deletion beginning at Chromosome 18 position 37,301,055 bp and ending after 37,303,313 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48934 | TCC CTT AAC TTC TTT CAG ATT CTT CC | Common | A | |||
| 48935 | CTG ACA GGA GAT GCT GGG ACT | Wild type Forward | A | |||
| 48936 | CAC CGC AGA AAT ACA ATG GA | Mutant Forward | A | |||
| 48937 | Fluorophore-1 | ACT CCT GAA TCC GGT GAT ACC | Quencher-1 | WT Probe | ||
| 48938 | Fluorophore-2 | TGT TTT TCT GGG TGG GTC TCT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48934 | 0.40 uM |
| 48935 | 0.40 uM |
| 48936 | 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.