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Stock No: 018431
Protocol 22066: End Point Analysis Assay - Trp53<tm3Att>
Version 3.0
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 = CAGTCT
Wild type = TTTTTT
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 17343 | CAT CAC CTC ACT GCA TGG AC | Forward | A | |||
| 17344 | GAC ACT CGG AGG GCT TCA CT | Reverse | A | |||
| 17347 | Fluorophore-1 | TGA GGA GTT TTT TGA AGG CC | Quencher-1 | WT Probe | ||
| 17348 | Fluorophore-2 | TGA GGA GCA GTC TGA AGG CC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 17343 | 0.40 uM |
| 17344 | 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.