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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
>chr15:85132604+85132707 104bp GGTGGGATCTACACCAGCAC TTCCTATATACACACACTCCCAACA
Mutant= 120 bp
Wild Type = 104 bp
Sequence
Wt Sequence: CTTGCAGTAGTGAAATAGGCAGACTTTAAAACACAGAAAATGTGGTGGGATCTACACCAGCACTGACTGGAGCCTGGAGGGGAAATCAgagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacgGCCTCTAAAATATCAGCTATCTCTTAGGGCCTCATGTCTTCCTCTTACAGCTGAGAAGAACAGACACACACACACACACACACACACACACACACACACACACGT
Deleted Sequence: gagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacg
345 bp deletion beginning at Chromosome 15 positive strand position 85,132,649 bp and ending after 85,132,993 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37483 | GGT GGG ATC TAC ACC AGC AC | Common | A | |||
| 37484 | TTC CTA TAT ACA CAC ACT CCC AAC A | Wild type Reverse | A | |||
| 37485 | TGT GTG TGT GTC TGT TCT TCT CA | Mutant Reverse | A | |||
| 37486 | Fluorophore-1 | AGG GCC TCA TGT CTT CCT CT | Quencher-1 | MUT Probe | ||
| 37487 | Fluorophore-2 | AGA GGG CTT CAC AGA GTA GTG GT | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37483 | 0.40 uM |
| 37484 | 0.40 uM |
| 37485 | 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.