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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
Mutant= 117 bp
Wild Type = 119 bp
Fam=Mut
Hex=Wt
chr3:96597447+96597565 119bp CCGGCTCCATCGGTGTTT TCCTCCCTCCAGAAGCAA
Sequence
Wt Sequence (deletions in lower case):
TAGGCTTCACTACCGAGACTTCAGGCCTCTCCCAACCACCACCCCCATACTCCTAGATATCTTCGGCAGGTCCCAGTCCCGGCTCCATCGGTGTTTTTGCCCGAGCCGCAGCTATGCTGCACACCGAgggccacgctcttcttcgagctgtgggacagggtaagctacgcttggcccgtttgcttctggagggaggagcctacgtgaatgagggtgatgcccagggggagactgcgctaatggcggcctgtagggcccgttacgacgaccctcagaacaaggcgcgcatggtacgctatctcttggagcaaggcgcggatcccaacatcgcagaccgtttaggacgtaccgcgctgatgcatgcttgcgccgggggtgggggagccgcagtggcctctctgctccttgcccacggcgcagacccttcagttcgagatcacgctggcgcctcggcgcttgtccacgccctggaccgaggggaccgcgagacccttgccacactgctggacgcctgcaaggccaagggcacggaggtcatcatcataacgacggatacctcgccctcgggcaccaagaagaccaggcagtatcttaattccccaccgtccccaggggtagaggaccctgcacccgctcctcctagtccaggggtctgcacatcgccttcagaagtccaactgcagactgcaggaggagggcgcgggttgttatcccctcgcgcccaggaagaagaggagaaaagagacatttttgaatttcctcttcccaagccccctgatgacccttccccttccgagcctctccccaagccacctcgacatcctccaaaaccactcaaaaggctcaattccgagccctggggccttgtggctcctcctcaaccggttcctcctgcagaagggaggcccggtttggagcgtctggctgctgaattcaatggcttgaccctgaccgggagaccgcgtctttctcggcgtcacagcaccgaaggcccggaggacccgcccccatgggcggagaaagtgacgggtgggggtcctctctcgcgccgaaacactgcgcccgaggctcaggagtctggtcttccttcagggctaaggcaaaaactgagccgcatggagccggtggagctcgacacccctggacatttttgccctgactcccccgagtccagccggttatccctggagcgccggcgatacagcgcctctccgctgaccctccctccagcgggctcagtttcctccccacgccagtcccaggaaagtctgcccggggctgtatccccgctgagcggacggaggcggagtccagggctgctagagagaaggggctccgggaccttgctcctggaccacatctcgcaaacacgaccaggtttcttgcctcctctcaacgtcagtccccaccctcccatccccgacattcgtccccaacccggaggtcgggcaccctcactgcctgcccctccaccctctggggcaccagggtctcccaggaccaagcgcaagttggtgagacgccactccatgcagactgagcagattcgcctgctaggggggtttcagagtctaggtgggcctgGGGAACCCGGGCGCTGAAGGAAATGAGGGGCGCCAAGGAGGGGGAGGGTCAGTACCCTGATAGAGGGAGAACCAGCTGATACCTCGGACACGGGATCTCTTGCATGTGCTC
This mutation is a 1457 bp deletion beginning at Chromosome 3 position 96,597,496 bp and ending after 96,598,952 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 66521 | TCC TCC CTC CAG AAG CAA | Wild type Reverse | A | |||
| 66522 | CCG GCT CCA TCG GTG TTT | Common | A | |||
| 66523 | TCC CTC TAT CAG GGT ACT GAC C | Mutant Reverse | A | |||
| 66524 | Fluorophore-1 | CTG CAC ACC GAG GGA ACC C | Quencher-1 | MUT Probe | ||
| 66525 | Fluorophore-2 | TCG AAG AAG AGC GTG GCC | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 66521 | 0.40 uM |
| 66522 | 0.40 uM |
| 66523 | 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.