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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
>chr11:115820986+115821119 134bp CCTGATAAAGCCGATTGTCC AGCCTTCTCCCACACCTCA
Mut = 120 bp
Wt = 134 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GAGTGAAGGTGGGTGACTAGGCCCTTGTGGCTGTTCCCAGGTACCCTTCCTTCTGCTATGGCTCTGCCTCTGACCTTTGCCTCATCCTCTTGGCCAAGCTCTCCCAAATCCCATCTACGgtggcggcagcctgagtgaacggacccgcctcctgttctcacccacagctctgtcgtgtcaccttacgagcggcagcttaatttggatggttatgcccagtgcctggaggatgggtctggcaagaggaagaggagttccagctgtcggtaatccacctgctgctgccacccctgggagcacatgggcttaagaaggggcctgaggacacagccgagggtatacctcagttccagtggaatgtacgtgtcttacggacagtaacaagggacaaatgtgctcttttttctcccgtgtctctaaggtctgttaataagaagcccaaggtcctgcagaactccctgccgcccgtcagcctggcagcctccagctcacctgcctgtgaccagagtagccaatacccagaggagaagtctcaggactcaagccccaggcagggctcagagctccccttgcagttcctggggtcctcagagccttgctctgatctggccagggaagacgtggggtgtgaccgagagagtcacaaaatagagaatggagctaagggaacccctaagctacgctggaactttgagcagatctcattccccaacatggcctctgatagtcgccacactttcctgcctgctccagccccagagctgctcccagccaatgtcattgggcgagggacagatgctgagtcctggtgccaaaagctgaaccagcggagggagaagctctcccgtagagaccgggaacagcaagcagtggtccagcagttccgggaggatgttaacgcagatcccgaggtgcggggctgctccagctggcaggagtacaaggagctgctgcagaggcgacagacgcagaagagccagccccggcctccacacctatggggccagtccgttacccccttgctcgaccctgataaagccgattgtccaggtatccctttaccctgagctatacccctataggctgttgggagcagagagagacttctgaaagtggatgcttagacgAGGGCTGGCTAAGGCCTGAGGTGTGGGAGAAGGCTGTGCAGTGGGGTGGGACCTATATAGGCTGTTCCGTGGGTGGTGGTGGCAGCCACTGGGGACAGTTTCCTGATGAGTCATTGACCAGTGATTCCCGTGGGGGATGAAAG
This mutation is a 1008 bp deletion beginning at Chromosome 11 position 115,820,077 bp and ending after 115,821,084 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 50627 | AGC CTT CTC CCA CAC CTC A | Common | A | |||
| 50628 | CCT GAT AAA GCC GAT TGT CC | Wild type Forward | A | |||
| 50629 | TCC CAG GTA CCC TTC CTT CT | Mutant Forward | A | |||
| 50630 | Fluorophore-1 | CCC TGA GCT ATA CCC CTA TAG GCT G | Quencher-1 | WT Probe | ||
| 50631 | Fluorophore-2 | TCC CAA ATC CCA TCT ACG AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 50627 | 0.40 uM |
| 50628 | 0.40 uM |
| 50629 | 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.