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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
Mut= 117 bp
Wt= 117 bp
Sequence
Wt Sequence:TGCCCTGACTCTAACATTTTCTGGCTTTTTCTTTTTTACTCTCCTTATAGGCTGAGAAGTTTGGAGACtCTTTcGTCTTtGAAGGCATGTTGAGCGAGCAAGTGAAAACGCATATCC
Mutant Sequence:TGCCCTGACTCTAACATTTTCTGGCTTTTTCTTTTTTACTCTCCTTATAGGCTGAGAAGTTTGGAGACcCTTTtGTCTTcGAAGGCATGTTGAGCGAGCAAGTGAAAACGCATATCC
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 38602 | TGC CCT GAC TCT AAC ATT TTC TG | Forward | A | |||
| 38603 | GGA TAT GCG TTT TCA CTT GCT | Reverse | A | |||
| 38607 | Fluorophore-1 | TGG AGA CTC TTT CGT CTT TGA A | Quencher-1 | WT Probe | ||
| 38608 | Fluorophore-2 | TGG AGA CCC TTT TGT CTT CG | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 38602 | 0.40 uM |
| 38603 | 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.