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Stock No: 002379
Protocol 21939: End Point Analysis Assay - Dmd<mdx-5Cv>
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 = T
Wild type = A
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 17920 | AGT TCA TTG ATG GAG ACG GAA G | Forward | A | |||
| 17921 | CAT GAG CAT GAA ACT GTT CTT TCA C | Reverse | A | |||
| 17922 | Fluorophore-1 | CGA GCA CAA GGA GAG ATT TCA AA | Quencher-1 | WT Probe | ||
| 17923 | Fluorophore-2 | CGA GCA CAA GGT GAG ATT TCA AAT | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 17920 | 0.40 uM |
| 17921 | 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.