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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
>chr3:159515386+159515508 123bp TCAGTCCTATACACACATACACACA TTTCCTGAAGGGGGAAAAATG
Mut= 140 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GACACTTTGTAACTCAACAGTATACTTTGATTTATACAATTACTAAGTATTAAAAACACTGCCTCAATAAAGAAACTGGAGATCCATCAAGAAAGACACCCAATGTCAACTTCAGTCCTATACACACATACACACACATACCAAACAAACAAATCTGTGTTAAAAGTGTTCAACattcctttcagtttgagtacaaactagaacaaaccaaagcatttttcccccttcaggaaaatacagaaaagataaatcatgaaagaataaccaatgaagatctagaaattgcacctgacaaccaagaaataagccaggaagatgttgaagaagtttggagatacgttattatgatatagtaagacaaaaataagcagtatttaagattctgaagtatgtggattattaacttgatttttttatccaaatatgcaaatgaataaaagtattacttttcaaaacttagaatttaaatatgcattatgatttttaataattgcttttggtacagatttagtacagtataaagaaagaagttataccttttaagtcatcactgtagtttgagatttttattggactcctctcaattttgttgagtaagtatatagtaaaagtaaatagtaaatataatccttgaattgttttaagagaaaaataaagtactcttatgtctttgtttgttgcaagtcatgttcttttcctcttattttataaagaaaaccattgtttatactctctttagcctgcaaaccattttaagtttgccatccatagaagaactcttaaacccaaaccaagtaattcctcaatatataatgtacaacatggccaacacaagtaagcatggagtggtcatactacaagacaaatcaggtaggtatttagaaaagaatctggaatcttttcaaacctagtaaccaaaggtgcATTTATAGGTTAGTATAGAAAGTAGAACATTTTCCTAGGACACATTCATTTAGCAATAATGATGGAATTTTTGGAAGTAAATATTATTTATGCTATCACAAAAAAGTATTTAATTGCTTAAATGAATTAATATGACTTTAAAAATATGGCAGTGATAAACA
This mutation is a 750 bp deletion beginning at Chromosome 3 position 159,515,449 bp and ending after 159,516,198 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46187 | TCA GTC CTA TAC ACA CAT ACA CAC A | Common | A | |||
| 46188 | TTT CCT GAA GGG GGA AAA ATG | Wild type Reverse | A | |||
| 46189 | CTT CCA AAA ATT CCA TCA TTA TTG C | Mutant Reverse | A | |||
| 46190 | Fluorophore-1 | CCT TTC AGT TTG AGT ACA AAC TAG AAC A | Quencher-1 | |||
| 46191 | Fluorophore-2 | AGA AAG TAG AAC ATT TTC CTA GGA CAC A | Quencher-2 |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46187 | 0.40 uM |
| 46188 | 0.40 uM |
| 46189 | 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.