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Stock No: 026096
Protocol 22042: End Point Analysis Assay - Otop3<m1J>-EP
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
Sequence
GCATGACCCTGGAGCCTCGGGAGACGGAGGCAG(g/a)GAAACTGTTCCC
TGCAGAATCCCCTAGGACCA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21146 | GAG CAG AGG AAC TCC AGC AC | Forward | A | |||
| 21147 | AAG AGG GGA CAA AGG GAT TG | Reverse | A | |||
| 21291 | Fluorophore-1 | CGG AGG CAG GGA AAC TGT T | Quencher-1 | WT Probe | ||
| 21292 | Fluorophore-2 | ACG GAG GCA GAG AAA CTG TTC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 21146 | 0.40 uM |
| 21147 | 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.