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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
>chr18:80912681-80912768 88bp TGATGTCCCCAGCGTTTG AAATGGGGTGGGATGAAACT
Mut= 97 bp
Wt= 88 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGGTGTGGGAGGAGGGATTTCTAAGGCTGGACCTGGGAGGAAGAGGACTGTGGGGCTGTTGTGATGGAGATGTAAAGTGAATAAAAAATAAATTATTGGGAAAAGAAGAATGGTGACTTTGTAATAGAATGTTCAAAGGCATAGGGTAATGATAGGTTAGCATTAGACAGAAAGATTAATGTCcaaggaatatagacaatcaatgttgattgctaactatgtagacttaggttagaccttttaaaaagcctatacaacctatacaatatatgtttatcttttgttttatgcagttgctgtgattggctaataaatgtttgtcaaaggaagaaagagctaaaagctcgaacagtatggcttggatgtccagagaagtgtgaagagaaacatcctaggaattctatcaaaaatcagaaatacaatgtatttacctttatacctggggtaagatgatcgcatcttcaatgtcagttgatgtccccagcgtttgccttctgatgacacatgtagaaactctgcttgttgataacactgtttggtagtttcatcccaccccatttcaaatactttatattttcttggttctgtctacttaagaaaatgcatacttttattattgctttcagttcatgataaacattcatgttataagaacataaaagtatggaaatagtaaagaacaggtaaacatattttagttctggggacatttgatatttgtttggagggaaaattagaataaatgttgaagcctgccaaggaatttcacaaaggaagtatacgatttgttggtcttcatgtgggctctctaacaagtggattgcatggctgggcctcagtaggagaggctatgcctactcctgctgggactagatgtcccagggtgggatggtacctaaggggggtggggtgggggcggtgctcccccttCTGTGAGAAGGGGAGGGGGGCGTGGGAGGAGGGATTTCTAAGGCTGGAACAAGATATATCTCTGTATTTATTTATAGACACCTTTTTATATCAAGCTGTTAATCTCATGCCTTGTCACATTGTTAACTCATTAGTTACTTGGAATACTCAAGTTTAAATATATTAATTAATTATTGTTTTTACTACTTTAAATTATATTATAAAACTAGGTTTATCATTATTTGGAATATTA
This mutation is a 748 bp deletion beginning at Chromosome 18 position 80,912,310 bp and ending after 80,913,057 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38886 | TGA TGT CCC CAG CGT TTG | Wild type Forward | A | |||
| 38887 | AAA TGG GGT GGG ATG AAA CT | Wild type Reverse | A | |||
| 38888 | GGC ATA GGG TAA TGA TAG GTT AGC A | Mutant Forward | A | |||
| 38889 | TTG TTC CAG CCT TAG AAA TCC | Mutant Reverse | A | |||
| 38890 | Fluorophore-1 | TGC TTG TTG ATA ACA CTG TTT GG | Quencher-1 | WT Probe | ||
| 38891 | Fluorophore-2 | AAT GTC TGT GAG AAG GGG AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38886 | 0.40 uM |
| 38887 | 0.40 uM |
| 38888 | 0.40 uM |
| 38889 | 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.