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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
>chr11:69001876-69001977 102bp TTGAAAGCAGGTGTCCTTGG TGGCAAGAATCAGTTGGGAAG
Mut= 117 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CGCATCTCAGTAAGCACTGTGCCATTGAACAGCTTTTCTTTTGTAACATGATGTGGCTAAGTTTCCCATACTGGGTTTCTGTATGGGTCTATGTACCTGAGTGCAGCGCCAATGGAGGCATCGGGGCCCCCTAGAACTAGAGTCTCAGGCAGGTGTGAGCCTGACATGGGTACAAGGAACCAAACTTGGAATTATTTCTCCATCTTCTGATTAATTTTTGAAAGCAGGTGTCCTTGGCATCCAGTCTGAAACCAGAGGCCAGTGgtgtgttgggtcgatagcagtgggcaagatctaccttcccaactgattcttgccaaggccttcagattataaccatctgactctttcctcaggctgaatttctcgactccagcatcgaccaacattgtttctgtgtgccaggcagccgggctgagggctgtggatcgggtagagaccacccgacgttaccgactttcggtaaggtgtgggcattgtggtaggagatcaagaggagcgagtgggccagagattagatcctgaactcgagcagagtcatgggagaagcagggggaaagctcttggcttttggcctgtcctgtatgcttaggaggtttggttgtttgctgcttgaaggtacctgtgaagggagaatcggccagcctgactaaccagacttatctgtgcatccttttagtctgaacaaagacgctGCATGGGTCTGAGTGTGCATAGCTCAGTGGTGGGAATTCACCTCCTATATACAAACCCTGGCCTTGATCTCTGCACTAGAACAGAAAAGTGGTGCAATGTGGTAGTAGTGAGGTGAATAGCTTGGAGGTCCAGAGCGAGTCCTTTTTCTCTTTTCTTCAGCCTTGTCTTTTGTTTTCTTATGACTGGATCTTCCCAACACCAGTTTACCGACCACCCTACAGCTGAAATGGAAGCCATCTCTCTGGCCGCCTTGCACGACCGGATGACAGAGCAGCACTACCCTGACCCCATCCAGAGCTTCT
This mutation is a 431 bp deletion beginning at Chromosome 11 position 69,001,500 bp and ending after 69,001,930 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 48017 | TTG AAA GCA GGT GTC CTT GG | Common | A | |||
| 48018 | TGG CAA GAA TCA GTT GGG AAG | Wild type Reverse | A | |||
| 48019 | AGA TCA AGG CCA GGG TTT GT | Mutant Reverse | A | |||
| 48020 | Fluorophore-1 | CGA TAG CAG TGG GCA AGA TC | Quencher-1 | WT Probe | ||
| 48021 | Fluorophore-2 | CAT AGC TCA GTG GTG GGA ATT C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 48017 | 0.40 uM |
| 48018 | 0.40 uM |
| 48019 | 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.