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Stock No: 010502
Protocol 8900: Standard PCR Assay - Ptrf<tm1Pfp>
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 = 157 bp
Heterozygote = 157 bp and 417 bp
Wild type = 417 bp
Heterozygote = 157 bp and 417 bp
Wild type = 417 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9183 | CTG TCC AGT CTC TCG CTC CT | Common | ||||
| 9184 | AAC TTG CTC ACG GTG TTG CT | Wild type Reverse | ||||
| 9185 | TCT GCA GAA TTC CAC CAC AC | Mutant Reverse |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTP KAPA | 0.20 |
| 9183 | 0.50 |
| 9184 | 0.50 |
| 9185 | 0.50 |
| Kapa 2G HS taq polym | 0.01 |
| EvaGreen | 1.00 |
| DNA | 0.00 |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -1.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 50.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
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.