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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
>chr3:137864556+137864710 155bp AGTATAAAGGGCGCGAGGA ACTCTTCACGTTACCATCTCG
Mut= 162 bp
Wt= 155 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGACGCCAGCGCGCGCTCCTCCCCAGCCTTCCAGCTCCTCCCTCCAGGCACGTGACACCACCGTCCACTCGCTCGGTGGCGCAGCGCTCTGTTGCTGCCCTCCCGCCGCGGCGCCCCTTCTTCTCCCGGTCCTCGTCCCGCCGCATCTCCTCCCCCCCTAACTCATCCCCCACGCGCCAATCATCGCTCGAGCTCCCGAGCGCCCGCCCCGCCACTCCGctgtgcgttctcccattggctggagcctcaaggacgcgtcccgagggcgggaggcaaccattggtgggccgaaccggccgagttcccggatgagggaacattctgcagtataaagggcgcgaggaaggcgggagacggcgcagtttgaatcgcggtccgacggaggagtgggcgctgggatctcgctgagcgtccgcctggcctcgtctcttcctcgctcgtcggagcttcagcacggtccgagatggtaacgtgaagagtgacggcgggcggccgcgggctgcaggctccgagcgggcggggccgccgtccccggggctgggagaggcggcggcggcggcgagtgggagcgtgcggggacggggtcgggacgagcggaggggaggtgttttcggagccccggagctccaggcgggaggcgacggttgggggggcgcgcacggcgcggctgggagccggcgcgggggcgcgcagccgcggcgggctcgcggcaggggtcggcgggggagaagggcgggcggggggcgagcggcggcccggggtccccggcggggcgggcggcgggcggccgagcgcgcgctcgctcgcctccaggacgcgctgtggtgggggaggggcgcgccgccttggtaattctatcttctccgtccctcccgggctcgcgggcgtcggcggcgccgcaggctggcggtaaggctggaaaggactccggaaaggccaagacaaaggcggtttcccgctcgcagcgagccggcttgcaggtgggtagcaactgccgcagtcgggtgaccgggcctttatgtttgcaccccccttgcttgtttgtctgcaccctccatgggtgttccatcttagcatgagtgatgtgtcgcgacttggccctgcggagacagtgaatactggggtttggggagggggggcgtcggatcacatgtgtgttaactggcggggcgcgttggtgcaccaccaccgtccttggctttggcgcgcacatcttatcttaacgaatttcctatctttgtgcattttttttcttgtttgttcgtttgtttgtgtgcataaaaatcaagttccctgtgggccgtattcatcgacacctgaaatctaggacaaccagccacggacgtgtgggcgcgaccgccgctgtgtacagcgcagccatcctggagtacctcaccgcagaggtaagcgggagtacgcctgtgatctggaaaagctctgggggggcgggggaaagtggcgagagaggaggaaagtgatgcaagaaaactcgcaggccctaagagcggcgcgcagaccgagggagcgctagagggagctcgcgttcagccgaggaggctgctgggccagagtgtgctgctcctgcaaatgcaattgcgtgccccttcggggttttttgggtggttttttttttttttttgcataattctgcataattttatcattcaggtagtagggaatgacaacagttggtaaataacaccattgaggttcaaagatcttgattgaaagtaagttggaatattgatcatcagatttttatttattttttgccttttaggtacttgagttggcaggaaatgcgtcaaaagacttaaaggtaaagcgtatcacccctcgtcacttgcagcttgctatacgtggagatgaagaattggattctctgatcaaagctaccattgctggtggtggtatgtcatccctaaactcctaacattctatttaagaagaaaattcctacactcccattgtttctagaagagatttcagtacagggtgctttcatggggtttggctttccacttagtttttgctaagatcattgttcatccctcaagttttcactttgtatatgaattttacctgctttgagttcatgtattgtttttttaaatgtttatccagaaggtagacttaatctgtgtccctctcctaggtgtcatcccacacatccacaaatcgctgatcgggaagaaaggacaacagaagactgtttaaggatgcctggattccttattatctcaggactctaaatattcctaacagctgtccagtgttggtgattccagtggactgtatctctgtgaaaaacacaattttgcctttttgtaattctatttgagcaagttggaggcttaattagccttccaaccaaccaaatttctgcattcgagtcttaaccatatttaagtgttactgtggcttcaaagaagctattgattctgaagtagtgggttttgattgagttgactgtttttaaaaaactgtttggattttaattgtgatgcagaagttatagtaacaagcatttggttttgtacagacattgtttccactctggtggataagctcaataaaggtcatatcccaaactagctttaaacttgcttaataatcgggacttaccttagatctcactcagcaacaagtacattctctgcttactaattaaacagtgcatctgtagtcataaccccgtggtacttttgttgtttctttgtatcatatgacctcttagggtcttaaaacttgggaagaagctggaggtaactgcttttggcgtcccccccccccccaattgacagttaactaatgtgaaagggcctgtatcctaaggtgggacacaagtacagagaccctcagggtacaacgaaagtacctccaCCGAATGAACTTCGGATATAATTTGCTTTTAGTGACCAGACACAGGTGTGAAATGCTAGTCACATCTTGATTCATGCCAAGCATGTTCCATTATTATACATTGACCCCTTTCCCCTCCCTGAGCCTTAAGACATGTATCCTAATTCCTGGAGCAAATCAAACAGAATAGGAGGTGCTTAAGCAGCCTATGTGGATGAAAGAACTTCCTGCCTTACAGCTG
This mutation is a 2664 bp deletion beginning at Chromosome 3 position 137,864,450 bp and ending after 137,867,113 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52793 | AGT ATA AAG GGC GCG AGG A | Wild type Forward | A | |||
| 52794 | ACT CTT CAC GTT ACC ATC TCG | Wild type Reverse | A | |||
| 52795 | TCT TCT CCC GGT CCT CGT | Mutant Forward | A | |||
| 52796 | GAC TAG CAT TTC ACA CCT GTG T | Mutant Reverse | A | |||
| 52797 | Fluorophore-1 | CGC AGT TTG AAT CGC GGT C | Quencher-1 | WT Probe | ||
| 52798 | Fluorophore-2 | CCA CTC CGC CGA ATG AAC TT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52793 | 0.40 uM |
| 52794 | 0.40 uM |
| 52795 | 0.40 uM |
| 52796 | 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.