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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
>chr9:106500294+106500384 91bp TGGAAGTCAAGGATGTGTGG AGAATAGCGAAGATGTTGGGAAG
Mut= 114 bp
Wt= 91 bp
Fam=Mut
Hex=Wt
SNP in WT Reverse Primer - rs263504341
Sequence
Wt Sequence (deletion in lower case):
ATAGAGCTCTCAGGACGAGGGGACCCTCTGATGCCTCAGGGTCTGGGAAGGAAGTAGCTGTGTGGTGGTGGCACTCATCTTTAATCCCAGACCTTGGGAGGCAGAGGTAGGCTGGTCTCTGAATTGGAGGCCAGCCTGGTCTACAGAGAAAGTTCCAGAACAGCCAGGGCTACACAGAGGGCTGGGAAAGAGAGAGTCTGGGAGCTCTGGAAGTCAAGGATGTGTGGATACAGAGCAgcgaggggagatggcatacacacacaattctaagccaacttcccaacatcttcgctattctattccataaacatgttccctgaatcagccccaggcacttggtctgagccgtagttttcccatcttcacactaacactgtctgctttcctgacttccccaattgctgagcacagggtgaggaacagcagaagcctgaagaacttaatgcacctactgacgatgctcctcaggagaagcagcagccagctgatctatcctcagaaacagagaaggcaaaggtaggaggagagtgggcctcggtgtctcctccgacccttgtcctttgcatggtccagagctcagatcctgggcaaatggtcctttattgccactctctcctgtttccttttcaggctaatactctggttcagacatggaaagcagacagtgttagtgtgaagttgggaagactatggctcataccaagggcctggggctgattccgggaacacgtttatggagtagagtagctaagatgttgggaagttggcttagaattgtgtgtgcatgttttatgattctgcacatatgtctgcttgtcaattaagatagaactgtgaaaattatcaggcttgatggtgcacaccaattgttccagtatgtgggaggctgagacacttaggaaagaggttgaggccaggaaaaattccatagactcaagacatacacacatgcacacatacatacacactcactcacacacatacacacatatacatatcacatacactcacatacatgcacacgctcacacactcacatacacacacacgctcacacacataaacatactcacacacgcatacacacacactcacatacataaactcacacactcatacatactcacaatcacacactcatacacacacacacccacacacatacatactcatacacacacatacacacatgatggggcagtgtgggaagagaagagagagaacggggaagagaagattggactgtatgagttgaaagaaatgtaacatgagggttcaacaagagggaggagaaggttgaggactggggagagtcaagagggacaaatgaacaaggacaccaattccccagaaggttctaatgggaagggacctggttctgtttaggtgggcaggctcagcctggcataaccatttcatatgtagaggcgcacaggttttgctaacctggtctatgggaaagctggaatgccaggcttgagtgagactcaggaacatggtgatctgctggtggcctgaagccagagggtgagggacagaggcaggacaccaacccaatcttctagtccctgccagtgtcctgcccaaggctatgaatttcaggggaaaagggaggggcagcgatggacaaggagtcagcgcccttttcagcatctccttgtgctgaggtggctgaggacactcactatgccttccctttttctttgcaagtccaagaaaaaacaggagctttctgaaaaggaccaagttgtaaagatccaagcttggtggcgcggtacactggtacgtcggtcgctgttgcacgcagccctcagcgcctggatcattcaatgctggtggcggttaatactgccaaagattatggagaagaggcggcagtccatgctggataccttccaacaggagcagtgggcagtcgtcaggttgcagtcctggatccgcatgtggcgtatccgcaggcgttactgccggttgcttaaggctgtccggaccatccagtctcactggagaggtcatacttgctcttcacggggtgttatcaagggccaataccgaatttcgaccagccagatgcatcttgagctggaggtcttgctgggttcggggccttgtattgtgaccgaatgtattcccctcccaataaaacagtgaggcggtggtcgtatgtggagtttctctctaacaaattccAGGAGGGCATGATGGGAAAGTTTTGGCATTTTGTATACTAGCTCTGGGGAAGTGGCAACCCTGGGTTCTTGTCTCCTGGCTTTTCATCCTCAGCTTCTGAGTCTAACTTGATATCTAAATGGATGCTGTCAAGAGAGTCCCACTAAATTCTTTTCTTCTCCTTCTCCTCCTTCTCCTTCTTCTCCTCCTCCTCCTCTTCCTCCTCCTCCTCTTCCTCCTCCTCCTTCCTCCTCCTTCC
This mutation is a 1953 bp deletion beginning at Chromosome 9 position 106,500,324 bp and ending after 106,502,276 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52800 | TGG AAG TCA AGG ATG TGT GG | Common | A | |||
| 52801 | AGA ATA GCG AAG ATG TTG GGA AG | Wild type Reverse | A | |||
| 52802 | AAA AGC CAG GAG ACA AGA ACC | Mutant Reverse | A | |||
| 52803 | Fluorophore-1 | CGA GGG GAG ATG GCA TAC A | Quencher-1 | WT Probe | ||
| 52804 | Fluorophore-2 | CAG AGC AAG GAG GGC ATG A | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52800 | 0.40 uM |
| 52801 | 0.40 uM |
| 52802 | 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.