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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
>chr9:107847034-107847158 125bp TCCAGAAGTTTCCTGTTGGTG CAGGCACTCAGAAGAACTGGA
Mutant= 92 bp
Wild Type = 125 bp
Sequence
Wt Sequence: GAACCTCATTCTTAACCCAGCTCTTATTCCTATGTATATTGTATTCAGTTTCTGTTCCTTTCTCTCTGTGGTGTGCTTCCCTCTGTTCATGGTGACATAAAGCCCGtgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacacATGGGCGATGAGGATGCGTGTTCCAGTTCTTCTGAGTGCCTGTGAGATATAAAGTGAGTGTCATAGCATCTATCCCTGTTTTATATGATTAAATTATTAAACCTAGTATTTATGTTCTTCACAATGGTGGTATGTTTACATTTGAGCCTA
Deleted Sequence: tgtgattcttggccagctgggattataggtttgtcattatgcctaatttcatttagcacattttgaattcttacagtgtatatggtttaattttgtcttcataaaaatttgtagatttagttatataaagtaaaaaccagtagggaaataaaaaattagcacttaacatgctttgtataattagtgtaaatgtcatatttttgcagattcttaatgcctttcgaacttctgatggcatacctgtaaaggacttgcagttgaaggagtataacacaggtgagttttttagatgtgcatagactcttggtttaggaagggtcttggagatatgtctgcatcatatgctactcaaaaatttgttttttaagaaatcacaagtatgatttccagaagtttcctgttggtgccttcaaataacaagcagccttagtcttaactgtgattctttgtatggctctttgtccacac
468 bp deletion beginning at Chromosome 9 negative strand position 107,847,543 bp and ending after 107,847,076 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37478 | TCC AGA AGT TTC CTG TTG GTG | Wild type Forward | A | |||
| 37479 | CAG GCA CTC AGA AGA ACT GGA | Common | A | |||
| 37480 | CCT TTC TCT CTG TGG TGT GC | Mutant Forward | A | |||
| 37481 | Fluorophore-1 | CAA GCA GCC TTA GTC TTA ACT GTG | Quencher-1 | WT Probe | ||
| 37482 | Fluorophore-2 | TCT GTT CAT GGT GAC ATA AAG CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37478 | 0.40 uM |
| 37479 | 0.40 uM |
| 37480 | 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.