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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
>chr3:54807127-54807276 150bp TACAAGAAGAAGAAGAGCGACCAG AGACCCTGAGTGTGTACCAAGT
Mut = 161 bp
Wt = 150 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GCTGGCCTCGAACTCAGAAATTTGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCATGCGCCACCACGACCGGCTGTCAGCCGGGTTCTTGGTCAGCCTGCACCTAGGAATCGCTTGCGGATGGCCGCCGCGCAGGCGCAGTCGGTTCTGTCTCGCACCTGCTCAACCCCGCCCCTCTCGCCGTTCCCAAGCAGAGGCTCGCGGACCGCTACGGCGCACGCGCAGTGCAGTGCTGGCGGTTCGGCTCCGGGACGTGCGGCGGGCTGCCGCTTGTCGAGTACGGTTGTGTTTCTCAAGAGCTGACGGGCCGCGGTGGCGGACCCGGGCTCGGGCGGCAGCACGGAGGCGCAGGCTGTGCccgagggctcggggccgagcaccgccagtccccggacggcccctccggtgacggtgctggtgatgcggcaggacgaggccgaggcggacggcgctctgaggccgggcctggcggggagtgaggcggcggcagacgccgaggacgaggcgggggacgacgacgccgacctgctggacacatcggacccggcgggcggcggcgagagtgcggccagcccggaggagctggaggacgaggacgcggagggtggcggcgcagcgcgcaggcgaggctccaagacctgcacgtacgagggctgccgcgagaccaccagccaggtggccaagcagcgcaagccctggatgtgcaagaagcaccgcaacaagatgtacaaggacaagtacaagaagaagaagagcgaccaggcactgggctctggcggcccgtccgcggccagcacaggcaacgtcaaacTGGAGGTATGCTTGCTCCTCGGGGCCCCGGGGCCAGGGCAGATGGGTGCCAACCTACTTGGTACACACTCAGGGTCTCCCCAAGGGCTCATCCTCTGCTCCAGCTGACATGGGCACTTTGGAGGTGATCTACACCTCTCCTTAGTAAACCCTTCCCAGTGTCTACCCCGAGGCATCCGGCACATTGACACACCCTGTTGGTTTTCTGTAACTTTGGTGACTTTGTTTTTTTTTCTTAGACTCCCTGGCCCCTGAGAAA
This mutation is a 453 bp deletion beginning at Chromosome 3 position 54,807,204 bp and ending after 54,807,656 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 49103 | AGA CCC TGA GTG TGT ACC AAG T | Common | A | |||
| 49104 | TAC AAG AAG AAG AAG AGC GAC CAG | Wild type Forward | A | |||
| 49105 | CGA GTA CGG TTG TGT TTC TCA | Mutant Forward | A | |||
| 49106 | Fluorophore-1 | CTC TGG CGG CCC GTC | Quencher-1 | WT Probe | ||
| 50046 | Fluorophore-2 | CGC AGG CTG TGC TGG AG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 49103 | 0.40 uM |
| 49104 | 0.40 uM |
| 49105 | 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.