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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
>chr4:43424380+43424477 98bp GTGTCTGCTCCTTGGGATG CATGGCCTCTGAAGTGGAG
Mut= 99 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
ACTCTAATTGAAGGCTGTAGTTGGAGCGACTCTGGGCAAGCATGTGGGTGACAGAAGACAAGAATTGGCAGGAGCATGCTGGTCAGTAGGTTAGTGACCAGTAGTCCAGGAGCTActacagggtaaataaccaagtgggccaaggggcaggagcttggccaagaaggcggagttgcttgtagagccaaacaaaacgcaaaggtcagagagggaaagctgtccatgaagagagcagccaacccccggaaaaggaaagacccacgagaaccgtcggtggggaggggcaacttcctcttccccactcaggtgtccgcctccaccaggtgggctagcatctccccaaggggacctgtgacagccacttctccctcacagggctggtaaaagctgtcaacaccgctgtggaccttattgtggcccattttggcacgagccgggatcctggcgtgaaggtatgctgacactatcaagagctgagctatgccctgcagactcggccgtcacctccctctgctctcggggacggaattcggggacagaaatggccgggggacatccaagagggtctgactaagccagactaaggtgaggcttctcacggttgccctatgtatccgcaggcaaagctgggcaacagttctgtgagccccaatgtcggccacctggttctgaagtacttgtgccctgcagtccaggccgtgctcgaggacggactcaaggccttcgtactggatgtgatcatcggccaacgtaaaaacatgccttggagtgtggtcgaggcttccacacagctaggtaggtactagattgcagccctggactccggactgaatttggggttccagaagttagaaatgtgatctccaaacctgccttctccccaggcccatccaccaaggtcttgcatggcctctacaacaaagtcagccagttcccagaactcaccagtcacaccatgcgcttcaacgccttcatcctcggcctgctcaagtgagtgcagaatggcaagatgtaagcagccagccccaccatagctttgcttcctctcaggggctggctgcctggaaggagaccggaggggtgtctgctccttgggatgcctttaccccaccgaggtgcagCCTCTCGGCAGGCGCTGACACCTCCCACCATCAGGCCCCTCCACTTCAGAGGCCATGGCCTGGCCTGTTTAGAAAGAAGCTGGGTTTCTCAGTGTTCGGTGTCTGCAAACGGTGATGTCTCA
This mutation is a 1006 bp deletion beginning at Chromosome 4 position 43,423,415 bp and ending after 43,424,420 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 41624 | GTG TCT GCT CCT TGG GAT G | Wild type Forward | A | |||
| 41625 | CAT GGC CTC TGA AGT GGA G | Common | A | |||
| 41626 | GCA TGC TGG TCA GTA GGT TAG | Mutant Forward | A | |||
| 41627 | Fluorophore-1 | TTA CCC CAC CGA GGT GC | Quencher-1 | WT Probe | ||
| 41628 | Fluorophore-2 | AGC TAC CTC TCG GCA GGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 41624 | 0.40 uM |
| 41625 | 0.40 uM |
| 41626 | 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.