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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= 110 bp
Wild Type = 110 bp
Fam=Mut
Hex=Wt
>chr8:105710272+105710381 110bp CTGCATTGGGAAAGAGGTTG GTTCTCTGACACAAATCTGGTAGT
Sequence
Wt Sequence (deletions in lower case):
CGACCGCCCGTCTCTAGGCGCAGACTGTACCACCGCCCCGCGACTAGCCCACTGCCCAGAGGGGTTCGCCGCCCCTCCCCCGCCAAGCCCCAGCGAGGGCTCTCACTGGAGCAGTTTTACCGCCAACTGTTGCAAGCCGGTAGGAGTTGCTGTTGCTTGGCAACAAATGGCTTCCTGAGGTCAGGGGTCAAATGACTAAggcgaacagccttctggggtttgtagttccagatgaggagtgattctaagtctcgggtctcaacaggagccttacctctcaacctgtctcccctaccagcactgagtaccagtccagccccgctctgggcctgaggttttctctgatcaactgtcgagggatgcactgattcctcatagcctatggcctcagaaggggccaagaggcagccagaaacccagaatggggcagcagtagggttagcccaggttgcagagagccttgaggtgacttgggcatttcaaagctcgggagatggaggggtagggcaggagaggtgggactctaggtaacccaaccctctaactgccaggccaagccctacaagtagagaggcctaagctggttaccttcatattttgtggccaatatgtagttaggagacaccaccacccccccaaccaagggagatgtactccaattgcagcagctttgggctttgaataactgatccccacaatactttgcctggccatcggctcccaatgacctatgtgtatcttctagtgtccaggaacagaagagtgtctggtgccggcacatgagacctgcaggagcccaggcgaagacaagtgtcccgtggggcacagcttagagccagagctccaggaggaaggaataaaggtgggagaggaagggctcaatgcaggggtggaagcaggagaggagagaggacccaagcctacgtcatccatcgtgagaccagctcatgggcccaagagaaagtgagagggctttcggagggaagggctgggaaggtgggtcttatgcccggagattcctaaatccccattccttcaaggcagagattcttgtctttgaccaatacccctcactcccaactctagaacttgggggctggggggttggtatggatttgggacttctagatcttgcaaggagactgagctaccataaatgctgagcctctactgggcttggagcttcactgtctaagaaatggacagctaactagctctagtggacaggcaggggtaagccctaggctcaagatataggaaccaaagactgaagcctgcctctttcaggcctcttgtcccaggtttgggttccccagccctcccagggccaagcaccatgtccatcctaggtcagaagttgagctgccaccgggggtgctgcagaagaaggaggagccagaaggcagccacagtgaatcctcactgtcctcgaaacagcacaaaaaagccaagaaacgcaagagtggcggggctcctgtgccgcccgctgtggccagcgcatccgcacccgccgccgagaccttggggttggagcgtgagtggcagtccctgggctcttctcttgttttctttgtcttctgaggctgacatggcacagcctggtgttctagcctttgctctgccttgggttcttgcaggaaaagcccagcgcctgcggccactgtaccaatacatcaactactgcaacccagagctgaatcaggaggaggacggggacagggagcccgaggtagagcctgaggccgagttagcactggttcccgaggagccgggcgtggaacagctgcagctgcagaccttgctgcccgtggcaggtgagctgggcttaggccttgctttaccctgccctaatccgttagtgccactgacccataatcttcctcctctggtagaggaggttggagaggagcctgggggtctgtctagtctgagggtgagtggcagcctcaaggctgaggtggataagacgacgcaagtggatatcgacaagatgttgagtgtctgtgctgcccctcttgtgccccctctatcccctcagtacaagtgaccatctagccactgagaatacccctggtctaggcctaaaccatggctctaagtccatcggcctaagccatcaggtggaggaaggaattttagtctttgtctacacttgagaatttgcgctcactgaaaataaacatttccccccttttagaatgtgattcttcaaatatgatgcctgcattgggaaagaggttggggagcagggaggaggatggctggtcgtgtggatcagtgGGGACCTTGAATGACGGATTCCTGAGTACTACCAGATTTGTGTCAGAGAACATGTCTACTCACCCCAGATTGGGAGTCCAAGGCAGACCAAAGTACACAGACCACCAAAGTCTGACTCCGTGAAACAATGGGTTTTAATGGGGTGACTTTTTTTTTTTTTTTTTGAGACATGGTTTCTCTGTATAGCCCTGGCTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGCC
This mutation is a 2081 bp deletion beginning at Chromosome 8 position 105,708,250 bp and ending after 105,710,330 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 62603 | CTG CAT TGG GAA AGA GGT TG | Wild type Forward | A | |||
| 62604 | GTT CTC TGA CAC AAA TCT GGT AGT | Common | A | |||
| 62605 | TAG GAG TTG CTG TTG CTT GG | Mutant Forward | A | |||
| 62606 | Fluorophore-1 | TGG CTG GTC GTG TGG ATC | Quencher-1 | WT Probe | ||
| 62607 | Fluorophore-2 | TCA AAT GAC TAA GGG ACC TTG AAT G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 62603 | 0.40 uM |
| 62604 | 0.40 uM |
| 62605 | 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.