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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
>chr4:139607713+139607822 110bp ACTGATGAGATGAAGCGCATGT CCAAACTCTTTCAGAACAGGTG
Mut= 117 bp
Wt= 110 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletion in lower case):
TGGGGCTGCTTTTTCTGGTGATGGGTGAGCTGAGCTGGCCCAGTCACCAGGCAGGACTTGGTCCCTGTGTCTGTGTTCTCCACCTGCATGCCTCACACCTCACGCTTCCTGCCAATGCTGCCTGCTTCGTAGAAGACCTATGCAACTCCCTGCCGTGGTGGCTGGTCCCAGCCATATCCGTCCACTCCTCgggacagtggttccctcccagacttaaccccctctgcaccccagccaagcacccccgttaccttggtctttccttccccgtccaggtggtccctaagaagaaagatcgtaaggtggcttcggatgaagacatctcggagcaggacggggaggtgaatcggttttcagatgaggaggtcggctccatgaacatcactgatgagatgaagcgcatgtttaaccagctgtgagtatccaggttgcccagccagGGGACATATCCTCAGCTAGGCTGAGACAAGTCACCTGTTCTGAAAGAGTTTGGAGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCACACTCGAGATCACACAAAAACCAGTTATCAGTCTGCGTGTCATTTCTCAGGC
This mutation is a 250 bp deletion beginning at Chromosome 4 position 139,607,520 bp and ending after 139,607,769 bp (GRCm38/mm10).
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 44131 | ACT GAT GAG ATG AAG CGC ATG T | Wild type Forward | A | |||
| 44132 | CCA AAC TCT TTC AGA ACA GGT G | Common | A | |||
| 44133 | TCG TAG AAG ACC TAT GCA ACT CC | Mutant Forward | A | |||
| 44134 | Fluorophore-1 | AGT ATC CAG GTT GCC CAG C | Quencher-1 | WT Probe | ||
| 44135 | Fluorophore-2 | CGT CCA CTC CTC GGG AC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 44131 | 0.40 uM |
| 44132 | 0.40 uM |
| 44133 | 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.