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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
>chr8:87567771+87567864 94bp AAACTTGGTTTCCCACTTGC AAGGCAGGATAAAGGGCTCTG
Mut= 90 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTTTGGGGATCTTGTCACAGATGCCAGGGATACATGCCAGGGTGGTCCAGAGGCCAAGGGACAGTGAAGGGACAAGAGGCTCTAGAGTATTGGTACTAAAACTTGGTTTCCCACTTGCCAGGAATGGCCTCCTATTTAGAGacagtgggtagaacccccacaggatggctgcagagccctttatcctgcctttacagaaaaagaaggaagagacaaagagtacagaagagaaggcaggaaagaaaaacgtaggcgctaagaagaagctagaaaaagtcagccgcaaagtgaccttccacaaggtaacacctctccccctcccccacagctgctcctctactatggtgtctgccacccctctctgaaccataatgacaccctaagagatgagtcttctcacgcgcgcgcacacacacacacacacacacacacacacacacacactccttgcagcttccccaacaacctctgctttcctgcagtccagtttaagggtgagcgtctcacattcctccacccacgtccctccatactccatgttaccagggcccagagaaccatgggaacactcagtcctgtacctacagggtatcccagacctgcctgcgagcatagactgcatgtaccctcatgagacaaggagaaataaaagtgaagtctcagggagccagcatggaagacacttctgctccctgaaactcttcctgggctcagaccaggtcctcgagcaagctggagaaatttctcagtttgggtttagaggatgctgagcaaggtcctctaagcagagcataacttctctcctgctttccaagggctacgtacgtcctgtcaagaagaaggaacccaccatttaccacaagagaaatgaaatagacctaaagagcaagcgtctgagcatgatcactgccaacttcagggatagcaataagaagtctggtaaggaaaatacacaggccctgggataccagaactgacctcccttgtccaatttctaagagataatggaagagctgcagcatccatgctgatccaacctgtgtacataggagccaggagccagcatgcagggttgttagggaatcacccagGGTAGCATTTGACAATCTGGGTATGCTGTGGATAGGAGGGGAGTGGTTGTTGATCTCTACATTGTCAGGAATCCAGCACAGTGTGATACATGCTCCTGAACTATTCCAGTGATGTCCAAATTCTTTAACTCAAACACTAT
This mutation is a 950 bp deletion beginning at Chromosome 8 position 87,567,814 bp and ending after 87,568,763 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 45262 | AAA CTT GGT TTC CCA CTT GC | Common | A | |||
| 45263 | AAG GCA GGA TAA AGG GCT CTG | Wild type Reverse | A | |||
| 45264 | ACC ACT CCC CTC CTA TCC AC | Mutant Reverse | A | |||
| 45265 | Fluorophore-1 | CAG TGG GTA GAA CCC CCA C | Quencher-1 | WT Probe | ||
| 45266 | Fluorophore-2 | AGG GTA GCA TTT GAC AAT CTG G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 45262 | 0.40 uM |
| 45263 | 0.40 uM |
| 45264 | 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.