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Stock No: 023451
Protocol 28412: Standard PCR Assay - Gt(ROSA)26Sor<tm1(Luc)Kael>
Version 3.2
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 = 183 bp
Heterozygote = 183 bp and 410 bp
Wild type = 410 bp
Heterozygote = 183 bp and 410 bp
Wild type = 410 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR3621 | CGT GAT CTG CAA CTC CAG TC | Wild type Forward | A | |||
| oIMR3869 | CCA GGG ATT TCA GTC GAT GT | Mutant | A | LUC | ||
| oIMR3870 | AAT CTG ACG CAG GCA GTT CT | Mutant | A | LUC | ||
| oIMR8546 | GGA GCG GGA GAA ATG GAT ATG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR3621 | 0.50 uM |
| oIMR3869 | 0.50 uM |
| oIMR3870 | 0.50 uM |
| oIMR8546 | 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.