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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
>chr8:121978231+121978363 133bp TTTTGGTTTTCAGGGTTTGG GCAATCTCACCTTTCCTGAG
Mut= 115 bp
Wt= 133 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGTTCCTGTGTGCGCCATGTACATGGTAGCTTGTGACAGGACTGGCCCTTGGGAGAGCAAAGCCATCCTGTTGAGCTTACCGAGCACATGGCCTGCTGCTGACTTCCAGCTTGCTAGTTGGAAACTGCTCCTCTTGTGGGCCACTAGGTGGCACACTTGATATTTAGAAGGTCCCTGGAAGGGGTCATTGACAGATTTCAGTGAGGTATAGCTGTGTCCAGCCATCTTTTTTTGGTTTTCAGGGTTTGGATAGTAGTCTTATCTTTCAGCATTAGAAAAATTTTGTAGCTTTACTGTataaatatttggatgagcatagtgacaagcttattactccatggcctcaggaaaggtgagattgcaagggcttattagacacagagggctgaatccctgtgcacggcccagggctcctcactgaatatgttggctcggcccagggctcactcagctgaaatacactgggtggacttaggagccagctagggtcctaaggacttgctgctcacagtggaagacatcctctctgaagcttttctgttttcatttccagtctttcctgtactctattaaccagacgatatgtttgcggttggatagcattgaggccaagctgcaagctctcgaggccacttgcaaatctctggaagagaagctagacctggtcaccaataaacagcacagtcccatccaggtccccatggtggcaggttccccacttggcgccacccagacctgcaacaaagtgcgatggtaagtccagaagaccgaggctccgcaggccaccctgggaaagcgctccgtgccagcccaggcgccgctgttactgatctgccctcgccccggATATCTGCCCTGGTGCTTCCATGGGGCTGGTTTCACTTAGACCGAAACCAAACACAGGCTCTGCTGGAACGGCCTGAGTTGCTCCAACTTTGTTTCTTGGCTTAAGCAGCTGGACTCATCTTTCCTTGCACCTGGCCATGAACTTGGCCCATGTATGTTGCTTCTTTTCTCTTTCTATCTCTGGCTGACTATTGCCTATGTTTTCTGCGGGGGTAATATTTATAATTTTTACTTGATCATAGAAAACTTTCCGTTTAGTCTGAGACTTGCCTTGTGCCTAATTT
This mutation is a 549 bp deletion beginning at Chromosome 8 position 121,978,299 bp and ending after 121,978,847 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48288 | TTT TGG TTT TCA GGG TTT GG | Common | A | |||
| 48289 | GCA ATC TCA CCT TTC CTG AG | Wild type Reverse | A | |||
| 48290 | TTT CGG TCT AAG TGA AAC CAG | Mutant Reverse | A | |||
| 48291 | Fluorophore-1 | TTG GAT GAG CAT AGT GAC AAG C | Quencher-1 | WT Probe | ||
| 48292 | Fluorophore-2 | AGC TTT ACT GTA TAT CTG CCC TGG T | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48288 | 0.40 uM |
| 48289 | 0.40 uM |
| 48290 | 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.