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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
>chr4:43496017+43496117 101bp CCTTCCAGAATGTGAATGGAG AATGGAGTTTGAGCCTTGATG
Mut= 100 bp
Wt= 101 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TATTCCAACTACTATGAGGGAGGATTGTAAGTGCAAGGCCCATCTGGACAATTTAGCAAGACTCTACCTCAAAGGCAGCGAGGTATAGGGATGCTAAATAAGAAGGCTGGGGTAAGAGCAATCTTCCCgtccctactggagagtttcacattcccaggagtgctttagcagtctggccatgtgtgggcatcctgacgtttcacttcatgttctgggtaagcagctggactagagacaactgggattctctgaggtggatgcccagagctttctctatggctcctctcttcactggctctgaggtgctggggcctttgctgcagccttgtctttatctgcttccacaacagggcccagatgaggctgctgttctcagagaggaaaaaaacaagaagaaatcctcgcagtcaggtgggttgagcatttagtggtggtttctggcgatggagtagatcaggggactgcaggagtaaccatgccctttccctcactcccagagcaacagctggtgcagctgacacaacagctggcccagacggagcagcacctgaaccacctgatgacacagctggatcccctttttgaacagtgagaatttgtggggtgggtgaggtgggagccaaggagacagactgggcagccccctgagtgggtatttctacagggtgacgactttggttggaacccagagggaacttctagacacaaaactaaagaccatccaccaccttctacaagactgccagcctggcacaggagtggaagttccagaaccaggtaaaaggttgggaggtttctatggctggagagccatggctgtgaaactagagcaagttggtctctccttcacagaggccagcataccctttactgaggacctggggaaagaagaccaagaagctggaaacagtcaggcctgggaagagcccataacctggagcccagaaacacggaacctagctccttcctgggaggtggagcaggggctgaggagaaggtggcacaagacggtgacaaagggtccagcagtaaatggggaacaaccactgaaggtttagtaaaaagagtcccctcgtactgggtgttcctctgtgctttttccactccctgctacacacacacacacggtgcaaaagccaagcactcttcccttattaggaagagagagactgccttccagaatgtgaatggagaagacagagagagagccacagttctgcccttgtGTTTCTGAGGCTACCGAGATGCCAGCCATCAAGGCTCAAACTCCATTTTCTCGTGATTTAGACAGGAAGGCTTTTGCAAAAACAACCCTTTATTAAGATCCAGTGGGGAAATAAATTATAGAACAT
This mutation is a 1098 bp deletion beginning at Chromosome 4 position 43,494,973 bp and ending after 43,496,070 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45257 | CCT TCC AGA ATG TGA ATG GAG | Wild type Forward | A | |||
| 45258 | AAT GGA GTT TGA GCC TTG ATG | Common | A | |||
| 45259 | CAG CGA GGT ATA GGG ATG CT | Mutant Forward | A | |||
| 45260 | Fluorophore-1 | AGA GAG AGA GCC ACA GTT CTG C | Quencher-1 | WT Probe | ||
| 45261 | Fluorophore-2 | AGA GCA ATC TTC CCG TTT CTG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45257 | 0.40 uM |
| 45258 | 0.40 uM |
| 45259 | 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.