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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
>chr18:21086048+21086146 99bp AGCGCAATTCAATGTTGTTG AACAGGGTGTTTGCCTCACT
Mutant= 103 bp
Wild Type = 99 bp
Sequence
Wt Sequence: ccagagcagccatgttgctgggttgaaattcctttacagagcgcaattcaatgttgttgtgcagtgtgtccccccactGAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTAtcttctctaaatactctcaacttaatgttttaaaatgattatctttggagctc
Deleted Region: GAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTA
425 bp deletion beginning at Chromosome 18 positive strand position 21,086,087 bp and ending after 21,086,511 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37463 | Fluorophore-1 | TGT CCC CCC ACT TCT TCT C | Quencher-1 | MUT Probe | ||
| 37464 | AGC GCA ATT CAA TGT TGT TG | Common | A | |||
| 37465 | TGA GTT ATC ATG AGC TCC AAA G | Mutant Reverse | A | |||
| 37466 | AAC AGG GTG TTT GCC TCA CT | Wild type Reverse | A | |||
| 37467 | Fluorophore-2 | AGG GAA GGA CAT ATA AGG TAA GGA AG | Quencher-2 | WT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 37464 | 0.40 uM |
| 37465 | 0.40 uM |
| 37466 | 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.