For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 023968
Protocol 22726: Standard PCR Assay - Park7<tm1Shn>
Version 1.3
Notes
This assay may amplify an artifact band of just under 200 bp in length.
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 = 280 bp
Heterozygote = 280 bp and 311 bp
Wild type = 311 bp
Heterozygote = 280 bp and 311 bp
Wild type = 311 bp
Separated by gel electrophoresis on a 3.0% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR7006 | GCT GAA ACT CTG CCA TGT GA | Forward | A | |||
| oIMR7007 | ACT TGG AGT CCC CTC GTT TT | Wild type Reverse | A | |||
| oIMR8162 | TGG ATG TGG AAT GTG TGC GAG | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR7006 | 0.50 uM |
| oIMR7007 | 0.50 uM |
| oIMR8162 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 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.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.