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Notes
Mut = 122 bp
WT= 144 bp
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
Sequence
SEQ
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 33822 | TGC ACC TAA AGA CTT TGT TTG G | Mutant Forward | A | |||
| 33823 | ATG AGA GGA AGG TCA TCT TGG A | Wild type Forward | A | |||
| 33824 | Fluorophore-1 | TTG ATT TGT TTC TTT TGC ACA TC | Quencher-1 | MUT Probe | ||
| 33825 | Fluorophore-2 | CCC GTC AGC TGC CAG TTC | Quencher-2 | WT Probe | ||
| 33826 | TTC CAG GAC AGC CAG AGC TA | Common | A |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 33822 | 0.40 uM |
| 33823 | 0.40 uM |
| 33826 | 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.