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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
Mut= 80 bp
Wt= 86 bp
Sequence
Wt Sequence (deleted region in lower case)
GATGTTTCTGGCAGGAGGTTGCCTGGCTCTGTGAGGGTGAAATAACTCCCACTCCATTTGCCTTGCATGGAACTGTAGGTCTTCTCCGAGCTGGAGTCCTACGTCCTGGCACCAGCTCCTTCGCGTACAAGCCATTACTTTCTAAGATTGTGTTGCCCATCCTCCTAGATAGGGGGAATCAAACTCATAAAATGTCATGCACTTCCTTGGTTTGGGAGAGCCCTTAGCCTCCTAACACATGCACACATGCACACATGCACACACACACACACACACACACACACACATACACACTCCTGAAGAAATCCGACTCCTACTCCATCCCTGCTTACTGCCCCCTCGACCACCACCACCACAGACCACCCAGCCACATCTTTCTCTTGGGGACACCAGGCCTctgcatgatggaagtcaggccagcaactgcttctgggtacctggaaagtatggtttccatctgacattatgaatcgcgagtaaggggatcaaagtcaggaacttcttttggaatagagagctgggatgcctacagcaggcagggtgatgtaagtttaaatccaaataacatatgttacctctctacccctatgttgcttcttccctgccacagggaaaaacgccatctttcgggcggtggtctgtggggaacccagacctgaagtgcactggcagagcagcaaaggagacctcagcaactccgccaagtaccagatatcctctgctcccggtagagaggagcacgtgcttcaggtgagccctgcagctgtttctacccatggttcacggctctcttcccagtccctaaaattcttaatggctctggtcccctggtgaagaagagatattcccacagttgggactATTCATCCTCAACCCGTCATTCAAGGTCTCCTGAGCCAGGAGAAGCCCAGATGGATTTGTTGTCATCTCCCTAATAATCAGGAGACCACCTGGACCATACTGGACCACAGTCCCAGGCCTACAGCTTTGGCTTTGGAAAGCTGGTTACTTGCCTCTTACTGTCTCAATTTCTTCACCTATGGGATAGCATAATTATAGTATACACGTTAAAGAATTATAAAGATTAAAAGTTCATGGAAAGGGTCTAGTTAGTGGTTCTAAACCTGTGAGTGTGACCCCTTTGAGGGTCAACCAACCCTCTCACAGAGGTCACATATCAGATATTCTGTACATCATATCATTACATT
This mutation is a 464 bp deletion beginning at Chromosome 1 negative strand position 135,980,217 bp and ending after 135,979,754 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37258 | AGA CCA CCC AGC CAC ATC T | Common | A | |||
| 37259 | TCC AGG TAC CCA GAA GCA GT | Wild type Reverse | A | |||
| 37260 | CTG GCT CAG GAG ACC TTG A | Mutant Reverse | A | |||
| 37261 | Fluorophore-1 | TCT GCA TGA TGG AAG TCA GG | Quencher-1 | WT Probe | ||
| 37262 | Fluorophore-2 | TAT TCA TCC TCA ACC CGT CAT T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37258 | 0.40 uM |
| 37259 | 0.40 uM |
| 37260 | 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.