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Stock No: 010633
Protocol 29549: Standard PCR Assay - Gt(ROSA)26Sor<tm1(CAG-taulacZ)Bene>
Version 2.2
Notes
Melting curve analysis is done using a Roche Light Cycler 480.
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
Transgene Tm = 87°C +/- 1°C
Wild Tm =83°C +/- 1°C (peaks at 83 and 81)
Wild Tm =83°C +/- 1°C (peaks at 83 and 81)
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0040 | CGT GGC CTG ATT CAT TCC | Transgene Reverse | A | |||
| oIMR3054 | ATC CTC TGC ATG GTC AGG TC | Transgene Forward | A | |||
| oIMR3621 | CGT GAT CTG CAA CTC CAG TC | Wild type Forward | A | |||
| 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 |
| oIMR0040 | 0.50 uM |
| oIMR3054 | 0.50 uM |
| oIMR3621 | 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.
Strains Using This Protocol
This is the only strain that uses this protocol.