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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
>chr4:11579360+11579461 102bp GCATACATGCGTGGAAATGT GCTCCAGATAGGATTTAATGCAC
Mut = 91 bp
Wt = 102 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
TTGTAAACAAATAACACAGAACAGTTCCTCTTTTGTTTTTATTAAAGTGACTGCGGTGCTGTTTCAGCCCCTCTGCCAGCATACATGCGTGGAAATGTGATATTCTCACTAAATTAGCTcattttaagctgtggttcctgcttatctccagacctttgtgcattaaatcctatctggagctgctccctgtatacacaagtgtgttttgcttgtgctctcctgccttctgtgagaagctagctttatgtaggtcaggcttggcttaagcggctttcctgaacctgcgtaagtcagcttaagctggaggaattttaataagccctcccctgtaggcgtgggcctaaatgaggctagcctagttaagcctgatctttctgtttgtgaaaagagctgagcagagctgaaggctgcatggtggtttcagaaacggcctacttaattccaaaagaacaatggtaggaaaggctagatcttccaattttaccttatctgaaaagcttgatttgctaaaccttgtgaagccatatgtgaaaattctcgaagagcacactaataaaaattcagtaatagtggaaaagaatagatgttgggatgtcatagcagttaactataatgcgattggagtagaccgccctcctcgcacagctcagggcctacgcagcctttacaaaaggctcaaagaatatgccaaacaggagctattgcagcaaaaggaggcccagtctgagtttaaaagcagtgtttcggagcccaccaagcaagtcatggaaatgattccccagattgccagcttcggcctcataagagacaggagccacatacagaggtaagccttattttattttgttgtgggaattatggcgcctcctatctcttgggctctggcttagctgtgctagacatgtgcatagcgagcgtccccacaaggggagccgtgaacagtgtctttgttttgctttcttaaaaaaaaaaaaaaaaagtaaagctctgaggagttctctgttaggacagcttgcagctgctttccctccatCCTGCCGATTCCACAAGTAAAAGTTAAGGCAGAATTGATGGGATGAAGCTGTGACATCACTAAGTGAACTTTAAAGCCTTCATAGCGTAGAGTGTATAAAGTAATGGGTTTTACTATGACATTTTTA
This mutation is a 914 bp deletion beginning at Chromosome 4 position 11,579,401 bp and ending after 11,580,314 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50616 | GCA TAC ATG CGT GGA AAT GT | Common | A | |||
| 50617 | GCT CCA GAT AGG ATT TAA TGC AC | Wild type Reverse | A | |||
| 50618 | AGC TTC ATC CCA TCA ATT CTG | Mutant Reverse | A | |||
| 50619 | Fluorophore-1 | AGC TGT GGT TCC TGC TTA TCT CC | Quencher-1 | WT Probe | ||
| 50620 | Fluorophore-2 | TTA GCT CCT GCC GAT TCC AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50616 | 0.40 uM |
| 50617 | 0.40 uM |
| 50618 | 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.