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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
>chr2:122174482+122174582 101bp GGGAAGGAGCTATTGAGTCCTA CTCCACCCAAGTCCATCTC
Mut = 103 bp
Wt = 101 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
AAAAAAAAAAACCAAAAAAACAAAAACAAAAACAAACCAACAATTGAAAACAAAGCCTCTCATCCTACACCATTAAACTAGTTGGGAAAGAAGGAATGAACCTCCCAAAGGGCTTTGTGTACTAGGATTGAATGTCGATGCTAGGCAGCCATTTGTGTGaaattccccaaggtgtgtggtgtggtgtaagtccctctccccaaatgctagtctcagtagtaatcgtggtgatgattatggaggtttccaggacaacaagatacaactgcaacaaaacctttccttggagtttctaaagctacatcagtttctgcacaacaaagaaaaggacattctaaatgacctccgggatgaggggaaactcttgaatgaggagatggaggtgaatctgaaccagattcaggagcagtgtcttgttgccaaagacatgctggcgaccattcaggcacggatggaacagcagaactccttcgacttcctcacagtgagattctgaatccaattgattatggggacacttggaaggagggaagagaaactgggagtggggggtggaaggaaagcaaatcataaccaagctcaacccaacccaaccatgtcctaacctacccaacccaacccaatttaacccaacccaacccgactaactggagcgcttgtaggtccttggggagaaaaaggaagccatgtttactttatgatcagtggaaatttttactttcatcttccttcaaaacgtgataggacaatttcactaataatgctacatggatcagttctttcttacttccccatatagaccacagaagctttatttttcaaaaaatatgtctcttatagtgtttgtagagtttgggccttgcagaaattataaactgaaatatatctaaaaagctttttgtcaaacttttgctacctgaagtctcacttctagagatcagatgattggcgatcactccagcactcagaagtctagttacccttccccctcagatcgtttctgatcttttcttggcggagtatttggaagcactcacttctcaaagagttgatcttaactatgggcttcctggctcttctcttgtttcctatgagagctggaattatagaaaatcagaaggttaccatgtgagaggtcagtttatgctgggttagcctgggatgctgcttagcaaagtctgtagaggatgaaatgttgttggcgatggcctgaggaccatctgtacaggaagccagtattgatgatcaaacctggatggctttcagtataaccagagaagttcctagagatgatgaacaggcatctgacttctctatttcttcttttctcttctaggacatcacaaagctcatagagaggcaagtgtttcttcatggttctctctaccctccttcttctacccctggagaatgtctaaaactaaactgtttgtgtgtcccttacagcatggagaaaggaatgaagacaattgtacctagacagctcatcgccaaaaagctgagcctaggccgcttcaaaggtcctatccagtatatcatctggaaggaaatgcaggctattctctctccaggtatagtgggaaggagctattgagtcctatggcccctaatctatatttcatctgtggccagagttagttctgtagtttgagagAGGATGGAGATGGACTTGGGTGGAGTTTAGTCA
This mutation is a 1495 bp deletion beginning at Chromosome 2 position 122,173,063 bp and ending after 122,174,557 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 51442 | CTC CAC CCA AGT CCA TCT C | Common | A | |||
| 51443 | GGG AAG GAG CTA TTG AGT CCT A | Wild type Forward | A | |||
| 51444 | TTG GGA AAG AAG GAA TGA ACC | Mutant Forward | A | |||
| 51445 | Fluorophore-1 | CAT CTG TGG CCA GAG TTA GTT CTG | Quencher-1 | WT Probe | ||
| 51446 | Fluorophore-2 | AGG CAG CCA TTT GTG TGA GG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 51442 | 0.40 uM |
| 51443 | 0.40 uM |
| 51444 | 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.