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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
>chr7:19525099+19525241 143bp GCTATCTGACCCACCTACTTCC GCGTCTGCCCTTCATCATC
Mut= 141 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CATTCATCTGTTTCTCTATCCATTAATATTCTTGTACCGGGTCATTTCATTCATTCCTGGAGTGGCCACAACTTAAGGCCCTGTCTTTCTTGTGACATCCTGGCTGCCTCACCCTCTTGCCTGGTCCTGGCTCTTTCTACCTCCCTCCCAtcctttctcagggaggtccctacttttgtcctagacctccaggcctcttccttcccacctacatgcaccaccagcctctccccgacccgtctccactgacgccttgcctcctctcccccagcgctgatgcagcaggaggccgcgcagcgtgagagcgaggagctgcagcgcgtgcagtggcagccacgtcgcgtgcgcggctggggcgtcccgaagctgctgtggttcctggtgtttctgcagcccgtgatcacggagctgcacctgcggcgcaggaatgtgcgcttcctgttcatccgcttcagcgcctggcagtacgcgggcaccgacaagctgtgggccgggctggtgaccacgctgtgtgagggcatccgccaccactacggcgcgctgccctttagcgtgtactcggtgctaggcaacaagccctgtggcccgcgtgatggcctctgccaacgcgagtggcactgccgacgtcgcgtgtgcctggcgctgctggcgctgctggccgcgctgtgcctgggtgtggggctgctgtacctgtcgctgggaggccacgcgccgggccacggagagcgtggtgtgctcaaagcgctgggcggcgcggccaccacgctatccggctcagggctgctcatggccgtgtactccgtgggcaagcatctgttcgtgagccagcgcaagaagatcgagcgcctggtgtcgcgtgagaagtttggcagccagttgggcttcatgtgcgaggtgaagaaggaggtggaactgctcacggatttcctgtgcttcctggagatctaccagcgacgccggctgcgtgtggtcctcgaggtcactgggttggacacgtgctacccagagcgcgtggtgggcgtcctaaacgccatcaacactctgctgtccgacagccacgctcccttcatcttcatcctggtggtggaccccagcattctggccgcgtgcctggagagcgcgggtaacatgaagggcacagcagacaacggctacctgttcctcaaccgcacagtcacgctacccttctccgtgccgattatgggccgtcgcaccaagttgcagttcctgcacgatgcggtgcgtagccgggacgatctgctgttccgggaattgacgatcaagctacagccgcagagccccgggaacttgggcgcaggtgagggtactcagctgctggcggtggagacgcagggggacgccgagcgcacacagggccgcgtggacgcggaggcggcgcggcgtatccaggaggcgctgtgctgcctgcatgacgagggtgattgcctgtatgagtacgtgcccgacaacgtggtgtccatgcggcgcattgtcaacacggtgcccatcaccgtgcgcctgttgcagcagcagcagcagcaacagcccgaccgcgtgggacccacaccgcgccacgcggtggcctgggtcgtcctcgctaaccagtggccttgtcgcctcagctgggtgctgcaatgcctggaggaccggcagcaggcagggggcgcgccagaggggcgctcgcgtctctgggatgtcttctgcgacaatagtcgcgagctgcacaccatgaccaaggccttgcagaacgtactggacctggatggcgaccctgagctttttgagcgctttctaggcactgatttccccttcaccgtggccgaggcgcagagtttgctgcgctgcacagtcaacctggaccattccatccgccgccgcatgggcctcatcagggccgtcagcgcgctcaagccgcccagcccacccaagtccccatcccaggacggtccccaggccagtcccagagccattatcgccgcaggcacgtcccatgcgggccaaggatcaggacacagtaaagaggcacatcagacccgggaccggacccatgggggcaagccaaggccgatggcctaagtccttaaatttcaggagcgtagaggctgtttgggtgtgggatggagaacaccagccacgggagacagcaacatccctcctgacctgatgagggtgtgagggcaagaggggctcaaaaccaatggggtatggccacacaccctgatgactttggaagccaaggggaagcaggtactactagcttcctggagtaggcagcttctacaccagtgtctgagtgagcagaggagtcagtgaggaacacagagtcaaagaagcaataaaggaagctgtggaaagccagggctgtctttcatgtcctggatttctggctaggtgagaatgctatctgacccacctacttcctcctcagggagccaggcctctggactgcaccctggcagctgaagttggcatcccAGGTGGTAGGAGGCTTGAGGGGGTGGTGGGTGAGGCTGCGGGGGCAGGGATGATGAAGGGCAGACGCCAAGTTGACTCAGGCAAAGGGGAGCACCCAGGCCTCAGGGGATGCCAGGCCTTTGTCCCCACACCCTAGCCTGCCCTCTGTGGGCAAAGCATGAACTGGCAC
This mutation is a 2325 bp deletion beginning at Chromosome 7 position 19,522,850 bp and ending after 19,525,174 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46192 | GCT ATC TGA CCC ACC TAC TTC C | Wild type Forward | A | |||
| 46193 | GCG TCT GCC CTT CAT CAT C | Common | A | |||
| 46194 | GGC CCT GTC TTT CTT GTG AC | Mutant Forward | A | |||
| 46195 | Fluorophore-1 | CAG GGA GCC AGG CCT CT | Quencher-1 | WT Probe | ||
| 46196 | Fluorophore-2 | CCT CCC TCC CAA GGT GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46192 | 0.40 uM |
| 46193 | 0.40 uM |
| 46194 | 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.