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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
>chr7:127083522+127083666 145bp AGAAGGGGTTGTTGGGTGTG TCTAACAGGAGGTGGGTCAGA
Mut= 157 bp
Wt= 145 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
GTAGTAGGATAGGATATTGTGGTATTGCTTCTATTATCTTAGGGTGGTGGCTCTGGTGCTCATTGTAATTAAGCATCAAAATGAATGCTCTGTTGTATGCTGTGGTTGAGTCAGAAAAGAAAGTCATATGGCAAGTGATGACAGAGGCATCTTTGTAGAAACTAGATCTAAAGAGGAGAAGGGGTTGTTGGGTGTGGGAGGCATCTCATCCcgagaccggattctctgcagggagtccctgggggccaagttctaccatctcccatgttggagaaccaaggccacatcaccacccaggcttctgacccacctcctgttagagctagcacctcccaccccctcactcatctccccacttctctccccagctcttcacggccgccccccaaggccccggcccctcccgtggctcagcctcccccctcatcatcctcttcgtcctcctcctcctcatctgcctcctcctcgtccgcgcagctcacccaccggcccccgacgccctcactgcccctgcctctgtccacccacagctttcctccccatgggttgcgcccaccaccaccccaccacccttccttggtctcccctggccccaccctgcccccacccccacctctgctgcaggtgccagggcaccctggggcctcagctgctaacgccctttctggtgagtttggggtcctggcagggtggagtggtggggggccatggccccaaggatgggctcagttggctttggcaggaaccaagggcctgtggagggagtggctcaaggctaaagggaaggccagtcaccttggcccaaagaggctgactcggtggcgccagatattaaagccttcttcccctcccctcccctcccacagagcaggacctgatcggccaggacctgaactcacgctacctgaatgcccagggtggccctgaggtggtgggagcagggggctcggcccggcccctggccttccagttccaccagcacaaccaccagcaccagcacacccaccagcacacccaccagcacttcactccctaccccccgggcctgctgcctccccacggccacatggtgagctcctccttgggctgctgctgaggcctaagggaaagtgtctcctacagggaaaggaagaccctggccccctggctgccagcttactcttcccttttcttccctagtttgagaaatacccaggaaagatggaaggccttttccgacataatgtgagtgtgtgtgagggtgtgtgaagtgtgggaatgtatgtatcagtggagtccagtcttcacctccaaaagcacgaaccctgagcttgagaaagctccttgggggtgttagggtggaagccaaagaactaatgggtagagcagacctgggctgcatctccactcccttcttggactagtgcccttctctccacagccgtacatggccttccctcccgctgtgcccgggctccctccgggtctcccaccagctgtctcctttggctccctgcagggggccttccagcccaaggtgagctcctaatccagacaactactacctcccatcccttataaacatcctggaaccctttacctgtttcccattccccaaaatgtttctagcccccattctcccttctggacccaagtttttgcaaagccacgatccctctcttgaccctttttccagcttagttctagacccctttttaacttggtactggctctgctgtctgatccctacactccccttcactcccctttcccagaacacgaaccctgagctgccaccacgactggggccagtgctgagcgggctaccccagaaggggacacaggtgaggggggcagggcaggtcctgggggagttggaaagtatgctgagggaaagcagaattgacgagagagggtgagttcttggttcttcccttaggtgtctaaagagagaacttttgtctaacaggtctaccccggCATGGGAGTAGAAGGTTGATGATATTAATAAGAAGAATGAAGTAATTAATAGAGCAGTAGTTTTGTATTTTAGTTTTCTTTGTTTGGTGTGATTCTAGATATCAAACCTAAGGCCTCACACATGCATCTACTACTGAGTTATAGCTCTAGCAAGACTGATTTTTTGA
This is a 1748 bp deletion beginning at Chromosome 7 position 127,083,557 bp and ending after 127,085,304 bp (GRCm39/mm39).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 58833 | AGA AGG GGT TGT TGG GTG TG | Common | A | |||
| 58834 | TCT AAC AGG AGG TGG GTC AGA | Wild type Reverse | A | |||
| 58835 | TGT GTG AGG CCT TAG GTT TG | Mutant Reverse | A | |||
| 58836 | Fluorophore-1 | CGG ATT CTC TGC AGG GAG TCC | Quencher-1 | WT Probe | ||
| 58837 | Fluorophore-2 | AGG CAT CTC ATC CCA TGG GAG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 58833 | 0.40 uM |
| 58834 | 0.40 uM |
| 58835 | 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.