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Stock No: 003248
Protocol 22873: Standard PCR Assay - Il12rb2<tm1Jm>
Version 2.3
Notes
This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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 = 500 bp
Heterozygote = 265 bp and 500 bp
Wild type = 265 bp
Heterozygote = 265 bp and 500 bp
Wild type = 265 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 |
|---|---|---|---|---|---|---|
| oIMR0204 | CAC GGG TAG CCA ACG CTA TGT C | Mutant Reverse | A | |||
| oIMR0221 | GCC CTG AAT GAA CTG CAG GAC G | Mutant Forward | A | |||
| oIMR0799 | GTG TGC AAG CTT GGC ACT GTG ACC GTC CAG | Wild type | A | |||
| oIMR0800 | GTT TAG CTT GCA GAC AAA CAA GGT CAT ACC | Wild type | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR0204 | 0.50 uM |
| oIMR0221 | 0.50 uM |
| oIMR0799 | 0.50 uM |
| oIMR0800 | 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.