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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
>chr1:165187875-165188017 143bp GCCTCTTTGCAGTGTTTTACA AGAGCATCCTTCTCCCTTCC
Mut= 131 bp
Wt= 143 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
AGCACCATTTTTTTTTAAAGATTTATTTATTATTATATGTAAGTACACTGTAGCTGTCTTCAGGTTACCAGAAGAGCCACCATGTGGTTGGTGGGAATTGAAATCAGGACCTTCGGAAGAGCCGTCAGTGCTCTTAACTGCTGAGCCATCTCTCCAGCCCCCTCGAAGCAACATTTTTATTGGCCCTTCAGaccatctctaacacaattgtgttttgtttgtttgttttcagtaagtgtgcatgacgtatgtaattgccgtttttgaaactgcaggttgtggaggcgtcgtcgctgagctggggcaccagaataaaaggcttcatcgcatgctttgctctgggaatcctgtgctcagtgctggtaaggtttcctcccacccgctgcctttgcccagctctcagctgtagccttccctttggcccagtgacttttctctgtgacactcattacatggagtggataagaaggggagggaggcagggagggcagctatgtggctctggtgggagggggtcactgtgctcaagtgcatgtgtcctctgcttccccttaggggactcttctgctgtgggtgcccaggaagggactcggcctctttgcagtgttttacaccctgggtaacatcatgtcaattgggaggtaacctttttcttttctctttgcttcaactgtgctgtttattaaacaataaaatatctaccgcgtttgagGGAGGAAGGGAGAAGGATGCTCTGTTGTTTTTAAGGATTCAGAAAGAAGGAAGGTAGATACTTGGCGTATATCACATTCATATCTTATTTCAAAAACATCTTATGTCGTGTGTTTTCTCTGTGACACCCCTGGAAGAAAGCAGAAATGGTGTCACCAGTGCTTTCTATGAGGGAA
This mutation is a 521 bp deletion beginning at Chromosome 1 position 165,187,898 bp and ending after 165,188,418 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 46139 | GCC TCT TTG CAG TGT TTT ACA | Wild type Forward | A | |||
| 46140 | AGA GCA TCC TTC TCC CTT CC | Common | A | |||
| 46141 | GTG GTT GGT GGG AAT TGA A | Mutant Forward | A | |||
| 46142 | Fluorophore-1 | ACA TCA TGT CAA TTG GGA GGT AAC | Quencher-1 | WT Probe | ||
| 46143 | Fluorophore-2 | TTA ACT GCT GAG CCA TCT CTC C | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 46139 | 0.40 uM |
| 46140 | 0.40 uM |
| 46141 | 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.