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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
>chr15:102147719+102147805 87bp GCCAGGAGAGACTGAATCCTT AGTCAGAGCAGAGACCAGCAG
Mutant= 97 bp
Wild Type = 87 bp
Sequence
Wt Sequence (deletions in lower case)
GCCAGGAGAGACTGAATCCTTCAGTCCCTCTCACTTTACTGGTAGAAGTGGACctaggttccgaggctgctggtctctgctctgactgatctctgatttcccctcccactcttcctgtggccacagaggagactacaaaggagagcaaaccccaaggaggtgcctcccgctctcgtgacacaaaccacagagaccggcagaagaatccacggacctctgctgcccctatacggcccaatcctgttcaagatagtgagatggtgcgtgggggggtggtagggaaggcagagggctggagatttccactggactcaaggaagtctggaccaggcaggctcctagaatcctgagtaccgccctcacccacccctgtccttctctagggcccctgccgcagacacttggattcagtactgcagcagctccagactgaggtcttccgaggcggagcacgtgggctctatgtgccaaactgtgacctcagaggcttctaccgaaagcagcaggtgagaagaagctgcctgtcctctgtgcaagctctgtgcgcggcagtgaggaagggtacttgatgcaaagctgcaaaaaggatgctggttctgctggagcatgtctccaaaaacaacagccaggtggtggagagaggcaggtggatctctgtgagttcgaggccagcctggtctatgtagtgagttccaggacagccggagctacacagagaaaccctgtctggaaaaaccaaaaccagccaaacagcaaccaaaaaaaggatggatacacacacacacacacacacacacacaaaactatttcttccaattttaagacccacatagatatgaaaaacatcaggaagattgtaacttcaagacttgcctaggctacaagtgaagggctggtaggaagacagagggctggagatttccacaggagatctttccatggacttgaggaagcctggaccaggcaggctcctagaattctgagtgagttcaagtccagcctgggaaatttcacgagcctctgtctcaaaacaaaatgtaaaatgaaaagaagtttatagctcgttggcaaagctctgcttagcagtactgtgggctccatctctagcgtgatctgaaagaaaaacaaaggccaacaattgcaatgggagacttaaagtctcttctaggataaggccttgtggtggcctcaaatgggagccctggggaatcacctggaggcgtctgagctggtgctggtggagccccttctgcattttgtgactcagtaggtctgggctagcctgtgaacacaagtttctaacataagtgatgttgggctgttgacagaaactgcattttgaaagctattgctctggcctagcagagtgggccctattcagtaggtaaagcaactaagagctctcagatgctttcctgctcaaagcctggacctcaccctgacctgcacctctctccccagtgtcgttcctcgcaggggaatcgccgtggcccctgctggtgtgtggatccgatgggccagcctttgccagtgtctccagatggtcaaggaagcactcagtgctctgccaggagcagtgggtgaagccccgagggagcctccaggacgccagcaggaacagggggctgtcatttgagctgtatgtgaagcaatgaataactctgtgccccaagacccaccttcacgccccaccccattgtccctgtcacccttgggcctttacacagctagttagaaagattgctgttggcttgtgtactaataaaacagtattgggggggtgtctctggcttacgtctctgcgtctgtctcattcagatctggggcctcgtgctcactgtctcccattggtcatagtgtttctaccttccctgcagagcacagccagcctcttccattgcccttggaatacagtagacTGCCACTGCCACAGGCAGCAAGCCCTTTCTTTATCCTCACCCGG
This mutation is a 1866 bp deletion beginning at Chromosome 15 position 102,147,772 bp and ending after 102,149,637 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62791 | GCC AGG AGA GAC TGA ATC CTT | Forward | A | |||
| 62792 | AGT CAG AGC AGA GAC CAG CAG | Wild type Reverse | A | |||
| 62793 | CCG GGT GAG GAT AAA GAA AG | Mutant Reverse | A | |||
| 62794 | Fluorophore-1 | TAG AAG TGG ACC TAG GTT CCG A | Quencher-1 | WT Probe | ||
| 62795 | Fluorophore-2 | AGA AGT GGA CTG CCA CTG CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62791 | 0.40 uM |
| 62792 | 0.40 uM |
| 62793 | 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.