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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
>chr5:129503672+129503759 88bp ATTGAATGGTTGACCAAAGC AGAGCTAAAGGCAGACAATCC
Mut= 84 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGAGAGTTCGGGACTAGCAGGAAGAAGCGAGGAAATTTCGCCCGTTTGGTTCTGTCGGATAGGTATTTATGGACTCGGTCTGTGTGAATGTAAGCTGTAATATTTAACTACTGAGAACAGATATTTTTTTTGCAAGTGGCATTGAATGGTTGACCAAAGCATACATGGTAAGAACTGCTAGcaagggggtacgttttctgctctaaaggattgtctgcctttagctctaagttctgttgtgctgtagagaggtgcttagattttgttatgattactccagtttaaaagtaggagagagatgactaggcatattaagtataaatctgaagctttgcatttgttttaatatttttatttcattagtttaattggtaagtatgtgcatagagtgtcacacacaaccagcatgggtgtagacttccctccagtgaccagcctgttcacattacagatacgacgggcgtggtcacctgcatgacctctctgcgtacgatgctgagtacgccacgtggaaccgagactaccagctgtctgaagaggaggctcgggtagaggcgctgtgtgatgaagagaggtatttagccttgcatacggacttgcttgaggaggaggcaaggcaaggtactgctcaagaaagacttacttcagccacacctttttaaaattttaagtatttaaaaagttactcccgttcatttttatactcactctttctgatattatcttgacagtaccCAGTGGATTGGAAAAACAGGAGTCTTTGCGTTCTGAGAGGACCTCAGGATAGTTTATATGTAGAGCCACAGAGAATTTTCCCAGCTTTTGAGGGCAGACTGGGATTTGAAAAAACACAAAAACAAAAAACCAAAAAACAAAACTCTTTAACTGTTCTTCTTTAACAGTGTT
This mutation is a 554 bp deletion beginning at Chromosome 5 position 129,503,713 bp and ending after 129,504,266 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44152 | ATT GAA TGG TTG ACC AAA GC | Common | A | |||
| 44153 | AGA GCT AAA GGC AGA CAA TCC | Wild type Reverse | A | |||
| 44154 | GGT CCT CTC AGA ACG CAA AG | Mutant Reverse | A | |||
| 44155 | Fluorophore-1 | AGG GGG TAC GTT TTC TGC TC | Quencher-1 | WT Probe | ||
| 44156 | Fluorophore-2 | CTG CTA GCA GTG GAT TGG AAA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44152 | 0.40 uM |
| 44153 | 0.40 uM |
| 44154 | 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.