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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
>chr16:94501187-94501291 105bp GCATTCAGCTTCTTGATGACTC GCTCACAGTTGACTCTGGATG
Mut= 100 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGGAACACTTTAAAACGGCCTTTTTCTTTTTCTTCTTTTGTGACCTGGGGTTGAGGGGGTGGCTGTCCTTAAGCACCCTTCTAACCCTGAGGCTTTTTACATTTCATGTCTTTCGGCCTGAGAGAGCTTTGTCAGCCAGTGTAGGTTGGGAGCTGATTCGGAGTGAGCTGTGATgtccgtttgtccctaatatgtgtacagggtggttgatgagtcagtctggtggccactgaaagatgaaatggctattattccttcttgttttagaaagaagtttccatggtttatagaacaattactcaacttggttttcagttcttatttgtaccagtgcaactggaataagtgacagccccctttccttctgtttctgatgtctttgtctctgttctccatagagagcttcacttcccaaattcttcattagaggacatctgaactccactaactgcgctatcacgcagcccctgacaggagagctggtggtggaacactcggatgctgctatccggagcatagagcttcagttggtccgggtggagacctgtggtaagacttctagagtatctaggtatctacacttgtactgtcagccgctaaaacaccccttcctgtactcttaacatggcagggcacatcacatcataacagagctattctgatggcccctgtggcctactcaccccaaaattcccacttgcagataatcactgaaatactaattaggtccccagcaccttaaatcttggaagcatggtaaaaccagtgtggtgatgtttgagaacatgcaaagtagaagagaaggtaactcccagtcctgaagaagaaattggagaagtgggtgaatgtgttgatcctggttggcacagtattgggcagtttggtgaccagtttgctttggtgtcatggtgtcctgaccccatgatgtttcctctttctccagggtgtgcagaaggctatgcacgagatgccacagagattcagaatattcaaatcgctgatggggacatttgtagaaacctttctgttcccctgtacatggtcttccccagactgttcacctgtccaacactggagaccaccaacttcaaagtgggtaagcagtccaggcctggcctgtggagtctaggaagggtctgggccacagggaagaagcgccttgcacgcaggctgcagctttttttttttttttttggtttttcgagacagggtttctctgtgtagccctggctgttctggaattcactttgtagtccaggctggcctcgaactcagaaatccgcctgcctctgcctcctgagtgctgggattaaaggcgtgcgccaccacgcctggcttagcattcagcttcttgatgactcaaaaaagggtaagaagctgcgtaagtagGTGTGTGTAGGCAGTAGAGCCTGCTGCAAGGCCCCATCCAGAGTCAACTGTGAGCCAAGCAGAAATACCGTGAGTCTATAGGAAAAGCCTTGACAGTTGTCAGGTAAGTAGTATGTGAGTAAATATCCAACAGGAAACAACTTAA
This mutation is a 1215 bp deletion beginning at Chromosome 16 position 94,501,242 bp and ending after 94,502,456 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44111 | GCA TTC AGC TTC TTG ATG ACT C | Wild type Forward | A | |||
| 44112 | GCT CAC AGT TGA CTC TGG ATG | Common | A | |||
| 44113 | TGT CAG CCA GTG TAG GTT GG | Mutant Forward | A | |||
| 44114 | Fluorophore-1 | AAA GGG TAA GAA GCT GCG TAA GT | Quencher-1 | WT Probe | ||
| 44115 | Fluorophore-2 | CGG AGT GAG CTG TGA TGT GT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44111 | 0.40 uM |
| 44112 | 0.40 uM |
| 44113 | 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.