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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
>chr18:35735985-35736089 105bp TCAGCTACATAACAACATGCTCAG TTGGGGTCAACTACACTCAGG
Mutant= 105 bp
Wild Type = 105 bp
Sequence
Wt Sequence: TCAGCTACATAACAACATGCTCAGTGGTGCAGGGAGCCGAAGACTGTACAtCCTCTTTCCATTGGACTGTGGGGTGCCTGACAACCTGAGTGTAGTTGACCCCAA
Mutant Sequence: TCAGCTACATAACAACATGCTCAGTGGTGCAGGGAGCCGAAGACTGTACAaCCTCTTTCCATTGGACTGTGGGGTGCCTGACAACCTGAGTGTAGTTGACCCCAA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 39817 | TCA GCT ACA TAA CAA CAT GCT CAG | Forward | A | |||
| 39818 | TTG GGG TCA ACT ACA CTC AGG | Reverse | A | |||
| 39819 | Fluorophore-1 | CGA AGA CTG TAC ATC CTC TTT CCA | Quencher-1 | WT Probe | ||
| 39820 | Fluorophore-2 | CGA AGA CTG TAC AAC CTC TTT CCA | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 39817 | 0.40 uM |
| 39818 | 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.