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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
Mutant= x bp
Wild Type = y bp
Sequence
Wt Sequence:
Mutant Sequence:
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 42609 | CCT CTC CCT GTC TGC TAA GTC | Wild type Forward | A | |||
| 42610 | GGA ACC CTG GGC ATT TCC | Wild type Reverse | A | |||
| 42611 | Fluorophore-1 | CCA TCC CTG CCT CTG ATC C | Quencher-1 | WT Probe | ||
| 42612 | TGG GAA TGG CAA AAT AGG A | Mutant Forward | A | |||
| 42613 | AGT GTA GCC CCC AAA GGA C | Mutant Reverse | A | |||
| 42614 | Fluorophore-2 | ATA AGC TTG CGG CCG GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 42609 | 0.40 uM |
| 42610 | 0.40 uM |
| 42612 | 0.40 uM |
| 42613 | 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.