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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
>chr4:97574907-97575029 123bp TTTGGAGGGGATGTAAGACG CAGGGTGTCAATCACCTACG
Mut= 118 bp
Wt= 123 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGTGATCTTGCAAACAGGAAGCGATTGAACTCACTGTGTTACATAGGAAGAGGTGAGCCATTTTTGTTGAATTTGGTTTCCTCTGAAGACTGAGCATTTTAAGAGAAGTTTGGAGGGGATGTAAGACGGCTGATTGTATGATaacgcaatggaaatgccactcttggaagttgagatcttaagtaattaatgtcataaatgtgtgtttaatcgtaggtgattgacaccctgccaggtgctcaggattccaaggggaaagacagtggcttgccctccaggtgcacactagctctcgctgaggaagcagggacaggagcaggcatgtactttgcatttggtaggaccagcgaatgtacatttgactctgtgttgtcacatgtcctttgtagtggcttcattagtaaaatagggatggtgttggccaacttacagggagtgctgtgaagattaactgaacctactattcaaaagaacaagaatactgggtacacacttagtaggcactcagcctggtttctgtttcctttatttattcttgggatcctagagctccttgcgtatctccttTGTAAATAAGAGGCTGTCATGTGATTTCTTTGGTGGCGAGGAGAATGGTCCTGTGGTATGTACTCCATTCAGATACCTCTCCAGGAAGACAGTAGCCAATATCTTGCAGCTGAACCTTCCTTCTATGGAAGAGAAGAGTTGTTTGTGCTTA
This mutation is a 425 bp deletion beginning at Chromosome 4 position 97,574,571 bp and ending after 97,574,995 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44476 | TTT GGA GGG GAT GTA AGA CG | Common | A | |||
| 44477 | CAG GGT GTC AAT CAC CTA CG | Wild type Reverse | A | |||
| 44478 | CTG GAG AGG TAT CTG AAT GGA G | Mutant Reverse | A | |||
| 44479 | Fluorophore-1 | ACG CAA TGG AAA TGC CAC | Quencher-1 | WT Probe | ||
| 44480 | Fluorophore-2 | CTT TGG TGG CGA GGA GAA T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44476 | 0.40 uM |
| 44477 | 0.40 uM |
| 44478 | 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.