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Notes
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 = TM2/TM2
Heterozygote = TM3/TM2
Wild type = TM3/TM3
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 18872 | GAA GGA GAT TCA TCC ATG CAC | Common | A | |||
| 18873 | AGC TCT GGG TAC TCC CTC TGA | Wild type | A | |||
| 18874 | TCC CCT AAG CTA TCA GGT TGA | Transgene | A | |||
| 18875 | TTG TTC CGA TTC AGT CAT TCC | Transgene | A | |||
| 18876 | Fluorophore-1 | TTG TCC TTA TCT GTG CAG AGA ACA | Quencher-1 | IC Probe | ||
| 18878 | Fluorophore-2 | CTT CCC TTT AGC TAG TTT CCT TCT C | Quencher-2 | WT Probe | ||
| 61925 | Fluorophore-3 | TGG TGA AAT TCT TTC ATC CTT CAT G | Quencher-3 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 18872 | 0.40 uM |
| 18873 | 0.40 uM |
| 18874 | 0.40 uM |
| 18875 | 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.