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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
>chr7:44460712+44460827 116bp CAGCCTTGGGTGCTTAATGA CATGCACCAATCGCTCACT
Mut = 100 bp
Wt = 116 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GCCAGCCTTGGGTGCTTAATGAAACCCTGTCTCCGAATACAAAgtaaaaagacagctgggaatgaaggataatggtagagcacctacctagaatcccccagtgagcgattggtgcatggctcagtggctattgaccatctagaatcctcagtcacagagtccctgtccagcatgtgagaggccctaggtcttgtccttaggcccacatactgatcgcagagtcgcgttcacctgctgtgaactggagcagcctgctgctcccacgcttgtcaggtgcactgacactggtctccccgcaggtgatccgcacggacacattcaagcacctccggcacctggagatcctgcagctgagcaagaacctggtgcgaaagatcgaggtgggagcgttcaatgggctgcccagtctcaacacactggaactcttcgacaaccggctgacaaccgtgcccacgcaggcctttgagtacctgtccaagctccgggagctgtggctgcgcaacaaccccatcgaaagcatcccatcctatgccttcaaccgtgtgccctcactgcgccgcctagacctgggcgagctgaagaggctggagtacatatctgaggcggccttcgaggggctggtgaacctgcgctacctcaacctgggcatgtgcaacctaaaggacatccccaacctcacagcacttgtgcgcctggaggagctggagctgtccgggaaccggctggacctaattcgccctggctccttccagggcctcaccagcctgcgcaagctgtggctgatgcatgcccaagtggccaccatcgagcgaaacgccttcgacgacctcaagtcgctggaggagctgaacctgtcccacaacaacctgatgtcgctgccgcacgaccttttcacgccccttcaccgcctggagcgtgtccacctgaaccacaacccctggcattgtaactgtgatgtgctgtggctcagctggtggctgaaggagacagtgcccagcaataccacatgctgcgcacgctgccacgcgcccgccggcctcaagggccgctacattggcgagctagaccagtcgcacttcacctgctatgccccggtcatcgtggagccacccacagacctcaatgtcaccgagggcatggcggctgagctcaagtgccgcacaggcacatccatgacgtcggtcaactggctgacacctaacggcacgctcatgacgcacggttcctatcgtgtgcgcatttcagtgctgcacgatggcacactcaacttcaccaatgtcaccgtgcaggacacgggccagtacacgtgcatggtgacaaactcggctggcaacactactgcctcggccacgcttaacgtgtcggccgtggaccccgtggctgcgggagggcccggtggcgggggcccagggggtgggggcggtgcagggggcgcgggcggatacacctacttcaccacggtgaccgtggagacgctggagacgcagccgggggaggaggcccagcagccccgaggtactgagaaggagcccccggggcccacgactgatggcgcgtggggcggcggccggcctgatgctgcagccccggcctcggcgtccaccacggcaccggccccgcgctcctcgcggcccacggagaaggctttcacggtgcccatcacggacgtgacggagaatgcgcttaaggacctagacgatgtcatgaagaccaccaaaatcatcatcggctgcttcgtggccatcaccttcatggccgccgtgatgctggtggccttctacaagctgcgcaagcagcaccagctgcacaagcaccacgggcccacgcgcactgtggagatcatcaacgtggaggacgagctgcccgccgcctccgctgtgtcggtggctgcggccgccgcggtggctggaggcgcgggcgtgggtggggacagccacctggcccttccggcgctggagcgagaccacctcaatcaccaccactacgttgcggccgccttcaaggcgcactacggcggcaacccgggaggcgggtgcggggccaaaggccccggcctcaactccatccacgaacctctgctcttcaagagcggctccaaggagaacgtgcaagagacacaaatctaaacccaggcgcacgggacgcgggacgcctgcccgagatgctcctgcttgccgagcaaccctggcggcgcctgcgggtcctgcaatgggaaaggacctggggaccaggagaggtggccccgccctccgccttgccgcgtcctctgccctcctgccctgcccgccaccttccaggggttgaggagcttcttcgaggggtacccttttctcctcaccccaacaaccctccttttatggctctttttttttcttttcttttcttttttttttttcttttgcagtttgacgcctgtccttccctcctactccttcaccctcaaaacacaagagacaggcaacttcaccagagcccagggggcaactccgacaatgcccccaacagcgagctccactcgcggccctggcaggcccaccccaccccacagactcttttgatactgaagggaggtttgcgtcaatcactacctgctctgtaattactttaaaaaaaaaaaaaaacacggaaaaagtaaaaaaaaatttttttgggggggggggcatgaaagtattgaagagaaacagggcacaaagctcctgagggtgggacgaggagaggataggcagccagcgatgctgtcctcccaggaggggagagtggatactccaaagttacagctctgggactctctatccccgagattccagagaggaacaggctcacccagcacatagcagcacggctccatctccccatGTGCTGGGGGTTCAGTAGCTGCCCTTCTGTGTCTCTGTCTCCATCCCCATGTCTAGCTGTTGTTCCTTTCTGACTCTGGTGCCTCCCCCATCACACACACAGCA
This mutation is a 2813 bp deletion beginning at Chromosome 7 position 44,460,753 bp and ending after 44,463,565 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53247 | CAG CCT TGG GTG CTT AAT GA | Common | A | |||
| 53248 | CAT GCA CCA ATC GCT CAC T | Wild type Reverse | A | |||
| 53249 | CAG CTA GAC ATG GGG ATG GA | Mutant Reverse | A | |||
| 53250 | Fluorophore-1 | CAG CTG GGA ATG AAG GAT AAT G | Quencher-1 | WT Probe | ||
| 53251 | Fluorophore-2 | CAA AGT GCT GGG GGT TCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 53247 | 0.40 uM |
| 53248 | 0.40 uM |
| 53249 | 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.