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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
>chr5:115238266+115238412 147bp TGGGCTTTGCAGGTACGAG GCACCTTCTGTTCAGTTTTCG
Mut= 147 bp
Wt= 147 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
CTAGAAAAACGCATTAAAAACCTTAACCCTCTGTTCTTCATTTTAAAACATAATCCTCTATTCTTAACAGAAAACAGAGGGCTGGGTGTGGTGGGACGCGTTGGTAATGCCAGCGCTTCGTAGGAAAAGGCAGGGGATCAGAAGTTCGGGGTCGTCTTGGCTACACAGTCAGTCTGGGGTTAGCTTGAGGAAAAAAAGAAACCAGGAAGAGAGGGAGGGAGGATACTATATTGTTTTTGGAAACCACTTGAAAAGAACTCCCCTCCacaaaactgtcgaaatcccctcaaagaattcgcgtatttggctgtatgggaaaacctgtgctctgacttttaggtcccagatcgtgcgacactctcggactttaagtgcagggctgaccttaaacgttcacttgggggactctgagcgcacagaacttgtgtggaaaatccccagcgccggccggaggcgaacgcatgcgctcttacgcccgccgcgggaggcggagcgtggagattgacagggcacgctgcctgtccgagcctagtgccgccttcctcccgcatccggcgcggacgccatggtgcggttcaagcacaggtaagcagcgctgtccccgcgccgtccccgcgacccgggccgcgctcaccccgctgtcgccgcaggtaccttctgtgcgagctggtgtccgaggacgcgcgctgccgtttgagcctggacgaccgcgtgctgggcgggctggtccgcgacacgatcgcccgggtgcacggggccttcggcgccgccgcctgctccgtgggctttgcaggtacgagggcgctgcggatgcagtgcggggcgcgctttgccagggcgaggcccggccagtgtgtcgcgttttgtacgatagtggAGGGTTCTAGAAAATCGGAGTGCGGCGGGTCGAAAACTGAACAGAAGGTGCCGATCGCGCAGTGGGACAAGGCAAAAAGTTCAATACCCAGCCCCCGGTGTGTGTGCCGGGGTGGGTTGAGTTGGGGGATTCCGTTTACTGCCCGTTTTAGTGGAAGATGGGTGTTGAATTCACTAGGCTAAATGAAAGTAGCCATGTAGAATTCCTAAGAGCCATGATTTGACAGGTCACGTTAGCACGTTTTAAAATGTCAAATGTATCCCTCAAAGTTTAAACCCGGCTGCGAGCTCGAACA
This mutation is a 611 bp deletion beginning at Chromosome 5 position 115,237,751 bp and ending after 115,238,361 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52786 | TGG GCT TTG CAG GTA CGA G | Wild type Forward | A | |||
| 52787 | GCA CCT TCT GTT CAG TTT TCG | Common | A | |||
| 52788 | AGT CTG GGG TTA GCT TGA GGA | Mutant Forward | A | |||
| 52789 | Fluorophore-1 | CAG TGT GTC GCG TTT TGT ACG | Quencher-1 | WT Probe | ||
| 52790 | Fluorophore-2 | AAG AAC TCC CCT CCA GGG TTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52786 | 0.40 uM |
| 52787 | 0.40 uM |
| 52788 | 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.