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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
Sequence
Wt Sequence (deletion in lower case):
ATGACAGATGGTTTTACTTTATAAATCAGTCTTCTGATTCTGTGTAGAATATAGCGTGACTTACATTATGTTTAAATGGATGCCCTGAAGAAAGCTGTGAGCCACTCTGTTATGATGGTCTAGCAGACAACCTGTGGGGAAGCAGAGGGTGGGGAAGCAGAGggtgcttgcctcggcataaagtgagcaaggtggacttgctcttctatagacccgtagcttccctgcctcgtgctggtttctcctgactgacttactcacaccaagtatctatcttttccagcctctagcccctgggtctggcacgatcaacatggagaaacctcaaaggtacccatcctggtacacagctagcagagacacgccacacactccctctgctaactccttgcttgattgatgggggacaccaaggaccagagagttcaaagtctttgcttaagggttgggggacagcagtgaccagaaccacttcctgccccctcatcctcccagaacccatgacacccgtgactcctctgggcgaatgagagagaagtgggaaagaccagagagatggaatccctgtgactgcacagggcctcctccctctgctcctgggggccttgcagggtttggactggagtcattttaccctggactcagtgtcctcctcagctttgggcaggactagatagcttgggaagaagatcctgaggaaactgtttatccttcactacccccagagcaagtataagccaaagcttgttatgaaatggcccacgatggccaaggaaaaggctcctgtgcacaacaaacgtagtctgatccttgtgtgctcgcttcttacagcatcaagaaccaaccacaggatgtatgcatgggaatcatgacaggtgagtcctgggtttccagccagactccaaatcgtccccaccccctgggacacagcagatgttttgttaataaagaagatgcactttattgcttcattgcatcactagtgactgggaagacgggaaggcatgctacggagacataagGGAGGCAGGGGATAGAAGACGTGTGACCACACGTCCTGAATAGAAAGCATGAGAAACATTCAGGGTAAGGGGGAACTGGAAGGACCAGACCAAAGCCTGTAAGATCTTGCTCCTGTATGACTACCTCAGAGGTGGATTCATGGCACTTACCTGAGAAAAATG
Mutation details
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 47032 | ACG AGG CAG GGA AGC TAC G | Wild type Reverse | A | Wt R | ||
| 47034 | Fluorophore-1 | AGC AAG GTG GAC TTG CTC TTC | Quencher-1 | Wild type Reverse | ||
| 49020 | CAG AGG GTG GGG AAG CAG | Common | A | Common | ||
| 49021 | ACC CTG AAT GTT TCT CAT GCT | Mutant Reverse | A | Mut R | ||
| 49022 | Fluorophore-2 | AGA AGA CGT GTG ACC ACA CGT CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 47032 | 0.40 uM |
| 49020 | 0.40 uM |
| 49021 | 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.