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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
>chr7:6131056-6131169 114bp TGCATCTTGATGGCTGCT CCATGGTAACTGCCAACACC
Mut= 123 bp
Wt= 114 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TCCTAGCCTGCAGACCTGGCCCTCCCATCCTCACTGGGTTTCAGGTGCCACCTGTGTTGTGCTTCTCATTGCCCTCCTACCCTTGGCCCTCGATAGCATTGGTCGGTAGACCTTTATGCAGGCCTGTAGCCTTAGGCATTGGTCTGCAGATCTAgagaaaggctaccagagacaacacccactcggaaggagatcaagtattagggagatggattatgaacacagaaatacttgaaatgccttgtgtggttggtgtcacacgggataaggtgtactggatatgcattggtacaacccagcagtccccaggttggaggaaccccgaaaggaagagctgccctgggccactcaataagctggaatccattggcagcctgcgcatggatgttcacctctgacttgcctcctccttccctcttgctttgcagtggacatcctcatcatggatgacgatgacgtccccagctggcccccgaccaagctgtccccaccccagtctgcacctcctcctgggcccccgcccaggccgcggccaccagcaccctacatctgcactgagtgtggcaaaagcttcagccactggtcgaagctgacacgacaccagcgcacgcacacaggcgagcggcccaatgcctgtaccgactgcggcaagaccttctcgcagagctcgcatttggtgcagcaccggcgcatccacacaggcgagaagccatatgcctgctcagagtgcggcaagcgctttagctggagctccaacctcatgcagcaccagcgcatccacacgggcgagaagccttatacttgccccgactgcggccgcagcttcacacagagcaagagcctagccaagcaccggcgctcccacagtggtttaaagcccttcgtgtgtccgcgctgcggccgtggcttcagccagcccaagagcctggcacgccacctgcggttgcacccagagctgtcagggccaggtgtggcagccaaggtgctggcagccagcgtgcggcgcgccaaggcaccggaggaggccacagcagcggacggcgaaatcgctatcccagtgggcgatggcgagggcattattgtggtgggcccgccaggggatggtgctgcagcggccgcagctctggctggggtggggacgcgggcaacggggacacggtcgcgcagggcgccggcgcccaagccgtatgtgtgcatggagtgcgggaagggttttgggcacggggctgggctgttggcccaccagcgcgctcagcacggggatgggctcggggtagcggttggtgaggagcctgcacacatctgcgtggagtgtggtgagggctttgtgcagggtgccgcactgcggagacacaagaagatccatgcagttggggcgccttcggtctgcagcagctgcggacagagtttctatcgggcgggtggagaggacgatggagaggaccagtcggctggtgcgcggtgtgccgaatgtcggggtggggaggcccggtagggacggggaggcccggggggtggacagggcccctcgggctcggtgggaactaccagtgtgcaagaccacgagggaggcccagagagaatcagaccgaaaggaacagcggccttgtctccttccttactccgtgctgttctgggtgtgaggggacctgcaactattgggttctcaggactgtccacagcctctctgccgacccgccctaccgcctggtgggctggccctttgtaggtttccattctgggagccagagactgagagaactgcaaggatcaattggcactagaccctcaagggaaagaccacccccactacaggtgttgggctgcggtgtcgtaagaaatggagggaatcctggccttatgcatcaaaagggcttccttcccttctagaattttcttttaatcatactgtctgacccgtcccatccattattttttctgccccccatgggtccttatcaattttctagcccaagtcgttcctcttcctcttggatttgcagtagcagaggcgtgaagtagcctcctgttcccagcacggtcttattgagaggtcctggcagtggaaaaagggatgtgggggcaataaatggcattattcaattgtcccagcactgcctgtttcttgagtttgggatggagaagggagagaggttaaacctagccatcctcactttggtgcacacaagtgacaagcaccagatgatgggatgaccatgttggggggcccagagtgtcccaagccagaaagagatgtgtttgccaaggagccccttgctgacctggtacaaggtcactgttgggaaggtagtctgttcctgctgggaaaaggatggcttatggaagacatagaaatagcacacccctctatctctggtccaagttctggtcactcatcagccatatatcagcattctctgcagagcaggctgcatcttgatggctgctactgttcagtgaaggggcggaggcggaggaggaggagGAGCTTGGAGTAGATTGTTTCTGGACCCAATACTGAAATGGTGTTGGCAGTTACCATGGCATGCTTTGTTCCAGATGCTCCTCAGTGCCAGTCCAGTGCATCCCCCAGAACCCTACAGCTGCAGGATCAGAAGGGACCTCCAGCGTTAGGTGTA
This mutation is a 2331 bp deletion beginning at Chromosome 7 position 6,131,115 bp and ending after 6,133,445 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42657 | TGC ATC TTG ATG GCT GCT | Wild type Forward | A | |||
| 42658 | CCA TGG TAA CTG CCA ACA CC | Common | A | |||
| 42659 | CGA TAG CAT TGG TCG GTA GA | Mutant Forward | A | |||
| 42660 | Fluorophore-1 | CTG TTC AGT GAA GGG GCG | Quencher-1 | WT Probe | ||
| 43740 | Fluorophore-2 | TGG TCT GCA GAT CTA GAG CTT GGA G | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42657 | 0.40 uM |
| 42658 | 0.40 uM |
| 42659 | 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.