For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 005023
Protocol 22633: Standard PCR Assay - Tg(TcraTcrb)8Rest
Version 1.3
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
Transgene (Tcra) = 600 bp
Transgene (Tcrb) = 500 bp
Transgene (Tcrb) = 500 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 |
|---|---|---|---|---|---|---|
| oIMR3388 | GGT CCT GTG GCT CCA GTT TAA T | Transgene | A | Tcra | ||
| oIMR3389 | CTG CTT AAC CTG TCC CTC ATG T | Transgene | A | Tcra | ||
| oIMR3390 | CTG GGC AGT GTT CTG TCT CC | Transgene | A | Tcrb | ||
| oIMR3391 | ACC ATG GTC ATC CAA CAC AG | Transgene | A | Tcrb |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR3388 | 0.50 uM |
| oIMR3389 | 0.50 uM |
| oIMR3390 | 0.50 uM |
| oIMR3391 | 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.