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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
>chr1:54550979-54551108 130bp TGACAAAGCGTTATCCAGCA TGCTTATAGCTTCCAGCATTACC
Mut = 142 bp
Wt = 130 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
CAGTGACAGATAGCCATTGTCAGGGATTATTTTAACTGATTGGTTAGAATTTCAACCATACGTATTTTATGCTTTTATGAAGGTTATACTCTTCAAGATCCATATAATTTAAGAATAATCCCctgcgtgaaaaacgataacttgttttgtgcattgggtatggtttatagcattcataatatttgtatactgtaccattttaaagttctttatgtttgcattgagtctgagttgtattaataaaagtgattaaaatggacccatttatttatttatttatttatttattttctgtagaaaatagagctcccaaagaaactgacaaagcgttatccagcatatgagttatatctttacggagaaggatcgtacgctgaagaacacaaaattctccctttgacaggaattcctgttctctttcttcctggtaatgctggaagctataagcaaggtaggtagtggaagaaagacgttaaagctgtgagactcagctccagacttgcaatcatgtgcgtaatgccagagtcaagtgctgtaagtgaaaagcttactctgtcaggtactgtacatgccacttcccaaaagaaagcaaacatttaagatagaaggaaaagaagagtcgaacactacttcgcagagagtgagaagaatgagatggtgtaggtgggcgaatagattatacaggcattcagaaatcagatggcatagggtagggggaatagattatagaggtactcAGAAGTAGAGGAAAGGTTAATGTTGGAAATGAAGTTTCTGAGAATGGAATCTGCAACATTCAGCGGGGAGAGAAATCTTAAATTATGTTGAACAAGGAAGAGATC
This mutation is a 615 bp deletion beginning at Chromosome 1 position 54,550,691 bp and ending after 54,551,305 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51391 | TGA CAA AGC GTT ATC CAG CA | Wild type Forward | A | |||
| 51392 | TGC TTA TAG CTT CCA GCA TTA CC | Wild type Reverse | A | |||
| 51393 | AGC CAT TGT CAG GGA TTA TTT TAA C | Mutant Forward | A | |||
| 51394 | CAT TTC CAA CAT TAA CCT TTC C | Mutant Reverse | A | |||
| 51395 | Fluorophore-1 | ACA AAA TTC TCC CTT TGA CAG GA | Quencher-1 | WT Probe | ||
| 51396 | Fluorophore-2 | ACC ATA CGT ATT TTA TGC TTT TAT GAA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51391 | 0.40 uM |
| 51392 | 0.40 uM |
| 51393 | 0.40 uM |
| 51394 | 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.