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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
>chr3:86906366-86906452 87bp TTCTGGGCTTAGGGGTTTTC CCTTTTCCTTTAACCTTCTCCA
Mut= 90 bp
Wt= 87 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
GGAAGTCCTTTATAATTTAGTTACTTGCTCTATTTAATTCTGCAGCCAAGGATACTGAGACTAGAAGTGTGACATAATGTCTTATCAAAAGTGACACATTTGGGTAAAATAAGCCCAGATCTCACTCTTAGTTTCTGGGCTTAGGGGTTTTCACATCTCAGAAAAGCAGTctacgtaattgagaagggagggaggtttggagaaggttaaaggaaaaggttgtaatgtcccttttcctgtgaccccccaaacttagggcttggcttgcaggtctctagaagctgttgtttacatataatggattcaggtctactatggccagtgctcagagatctgatattatgctaattattatttttgttttttaggtgaaagttacgtgtgtgcctccaacgaaccatttcgtaaagttgattacaccaaaaacgttaatccgaactggtctgtgaacatcaagggcggaaccacccgcaccctggccgtggcctctgcgaagagtgaggtgaaagaaagcaaagacttcattaaacccaagttagtgactgtgattcgaagcggagtgaagcctagaaaggccgtgcggatccttctgaataaaaagactgcccattcctttgagcaggtcttaacagatatcaccgaagccattaaactagactcaggcgtggtcaagaggctgtgcaccctggatggaaagcaggtaagtgcttctgcctgcagctcgattgttgattatccttgtgggtatttgaagagatgcctggaacttgctgtgtagtccaggctgaccttgaacttgggatcttcctggtcctgccaagtgctaggattagggtgtgcaccatgcccaactattgcagttagacattccacagaatgtcctggtctcctggaagtaatcctagtGGACTTTTTTTATGAAGGATTGTGTGTGTGTGTGTGTGCGTGAATTAGCCTTTAGCATAAAAAATATAAAGGGCTTCCGTTTCTCCAATTATCTAAGAGCCCATTATATGCCTCTTCAGTTTGTAAAAGGAAAAGATTGAATTCCTGGGGAATAAGTTGCAGTACTGTCCTATGCTAAG
This mutation is a 736 bp deletion beginning at Chromosome 3 position 86,905,679 bp and ending after 86,906,414 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44702 | TTC TGG GCT TAG GGG TTT TC | Common | A | |||
| 44703 | CCT TTT CCT TTA ACC TTC TCC A | Wild type Reverse | A | |||
| 44704 | AAG GCT AAT TCA CGC ACA CA | Mutant Reverse | A | |||
| 44705 | Fluorophore-1 | ACG TAA TTG AGA AGG GAG GGA G | Quencher-1 | WT Probe | ||
| 44706 | Fluorophore-2 | AGC AGT GGA CTT TTT TTA TGA AGG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44702 | 0.40 uM |
| 44703 | 0.40 uM |
| 44704 | 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.