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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
>chr7:4528094-4528212 119bp GGAAGTCCCAGCTTCAATCA GAAGGTCTCACTCCGCTCTG
Mut = 113 bp
Wt = 119 bp
Fam = Mut
Hex = Wt
Sequence
Wt Sequence (deletions in lower case):
GTTTTGGTTTGCTTTCAGATAGTTTTATTGTATTCCAGGGGCTATGTCACCCTTTTCTGATCTCTGAAAAATCTTGcccatggtgaacagatgtacacactcaggcacacacacacacacacacacacacacacacacacacacacacacaccgaataaaaagtttaatttattaaaagtatattttaaagacaagacttgagactatatgtcagcttgctttgcttttgggaagtcccagcttcaatcagcagtgctgggtggtggttgcagttcctggattggtgcagtttgtcaagtctccctccctgtttcatccttccctcctccagagcggagtgagaccttcctggagctgtgggggaacgcgctgctgcgaccctcggtagctgccttcctgcgcgctcaggctagccacctggctaatctggtcctggagcctgatcgcctggaggagcaactaccctggcttagtcttcggctgctcaaggtgccacagctgcccacctttgttggagggaagtggaatttgcttgaaatttgggtggctgcaggaagaatggaaatgagcagttcagaggaattgaggggctggagcaggctggcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtcctaattccacaattccacctctgtaactcccactactgtggcctagGGTTAGGGGAGACTTTGCTCTCTGTGCCTCATTTTTTCCGCCCCCATAATCACTACCACCTAATGGGATGGAAGGGGAGCCCTCAC
This mutation is a 617 bp deletion beginning at Chromosome 7 position 4,527,750 bp and ending after 4,528,366 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 52323 | GGA AGT CCC AGC TTC AAT CA | Wild type Forward | A | |||
| 52324 | GAA GGT CTC ACT CCG CTC TG | Wild type Reverse | A | |||
| 52325 | GGG GCT ATG TCA CCC TTT TC | Mutant Forward | A | |||
| 52326 | CTT CCA TCC CAT TAG GTG GT | Mutant Reverse | A | |||
| 52327 | Fluorophore-1 | CTG GGT GGT GGT TGC AGT T | Quencher-1 | WT Probe | ||
| 52328 | Fluorophore-2 | TTG GGT TAG GGG AGA CTT TGC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 52323 | 0.40 uM |
| 52324 | 0.40 uM |
| 52325 | 0.40 uM |
| 52326 | 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.