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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
>chr13:76056727-76056836 110bp TCCGGATTAAGGATGGAAGA CCTTGCTCAGAAGTCAGGTTG
Mutant= 90 bp
Wild Type = 110 bp
Sequence
Wt Sequence: TAACTTTGGGAAATCGCCGAGACCCAGCGGTGTGGCCCGCGCCACCTCTCATAGACTTTGGCCTTTTAAGCGGTATCTCTCTTTCCTCCGGATTAAGGATGGAAGATCctttcagcctcgcaattctgaatcccgcatccaacctttctgtacccactcagccaagctggagtctcaacctgacttctgagcaaggagcctctgtcccaggaccgcattcgcctccacgcgggccacccagccaccggatccatctggtcttcttggggatcatcctggtggccgcggtagctggcaacaccactgtgctctgccgcctgtgcggcggtagcagcggaccctggccgggtcccaagcgtcgcaagatggacttcctgcttgtgcagctagcagcggctgacctgtacgcaagtggaggaacagcattgtcgcagctggcttgggagctgctgggcgacccgagaccggctctgggcgacttggcttgtcgcctttcccatctgctgcaggcatcggggcggggcgcctccgcccacctggtggcgctcattgccttggagcgccagctcgcggtgcgcattcctcagggccctcagttgccagcacgtgccctggctgccctgagctggctgctggctctgctactggcgctgcctccgaccttcgtggtgcgctgggatgctcctccatcttcaaccgccaatgcctggccagggaagcattgctgcaggggcatcttcgcgcccctgccgcgctggcacctgcaggtctacgcgctctatgaagctatcgtgggcttcgcggcgccggtcgctctcttaggcttctcttgcggccacctgctgtgtgtctggtggcagcgtgggtctcaggctccggtggctaggatgccctggtcgcccagtatggccagagcctccttgcccagcgcgttgccccaagccaaggtgcaaagcttgaaaatgagcctggcgctggcactgctcttcgtgggctgcgacctgccctacttcgccgctcgcttggcagccgcctggtcatcgaagcccgcgggtgactgggagagagagagcctggtggcggcgatgcgcgtcctagaggtggccaacagcgccatcaaccctttgatctaccttttcttccaggcgggcgactgccgtctctggcggaggctgcggaggcgcctaggagttctgtgctgcgtgcgggaggaagaagccgatatcagcgaatgggctggggaccaccaggcactccaccgccatcgctggccccaccctcattatcaccatgcccggcgggaggagcggaaccagggctgcctgcgcccacccccgccacgccccagaccgccgccctgctcctgtgaaagtgcCTTCTAGGAACTGCTGAACGGACAGGTCCTTGGCCACTGTAGCACAGCCTTCAG
Mutant Sequence: ctttcagcctcgcaattctgaatcccgcatccaacctttctgtacccactcagccaagctggagtctcaacctgacttctgagcaaggagcctctgtcccaggaccgcattcgcctccacgcgggccacccagccaccggatccatctggtcttcttggggatcatcctggtggccgcggtagctggcaacaccactgtgctctgccgcctgtgcggcggtagcagcggaccctggccgggtcccaagcgtcgcaagatggacttcctgcttgtgcagctagcagcggctgacctgtacgcaagtggaggaacagcattgtcgcagctggcttgggagctgctgggcgacccgagaccggctctgggcgacttggcttgtcgcctttcccatctgctgcaggcatcggggcggggcgcctccgcccacctggtggcgctcattgccttggagcgccagctcgcggtgcgcattcctcagggccctcagttgccagcacgtgccctggctgccctgagctggctgctggctctgctactggcgctgcctccgaccttcgtggtgcgctgggatgctcctccatcttcaaccgccaatgcctggccagggaagcattgctgcaggggcatcttcgcgcccctgccgcgctggcacctgcaggtctacgcgctctatgaagctatcgtgggcttcgcggcgccggtcgctctcttaggcttctcttgcggccacctgctgtgtgtctggtggcagcgtgggtctcaggctccggtggctaggatgccctggtcgcccagtatggccagagcctccttgcccagcgcgttgccccaagccaaggtgcaaagcttgaaaatgagcctggcgctggcactgctcttcgtgggctgcgacctgccctacttcgccgctcgcttggcagccgcctggtcatcgaagcccgcgggtgactgggagagagagagcctggtggcggcgatgcgcgtcctagaggtggccaacagcgccatcaaccctttgatctaccttttcttccaggcgggcgactgccgtctctggcggaggctgcggaggcgcctaggagttctgtgctgcgtgcgggaggaagaagccgatatcagcgaatgggctggggaccaccaggcactccaccgccatcgctggccccaccctcattatcaccatgcccggcgggaggagcggaaccagggctgcctgcgcccacccccgccacgccccagaccgccgccctgctcctgtgaaagtgc
1267 bp deletion beginning at Chromosome 13 negative strand position 76,056,814 bp and ending after 76,055,548 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37622 | TCC GGA TTA AGG ATG GAA GA | Common | A | |||
| 37623 | CCT TGC TCA GAA GTC AGG TTG | Wild type Reverse | A | |||
| 37624 | ACA CCC TCC GTT GTC TGA AG | Mutant Reverse | A | |||
| 37625 | Fluorophore-1 | CTG AAT CCC GCA TCC AAC | Quencher-1 | WT Probe | ||
| 37626 | Fluorophore-2 | TGC TGA ACG GAC AGG TCC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37622 | 0.40 uM |
| 37623 | 0.40 uM |
| 37624 | 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.