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Stock No: 002597
Protocol 27805: Standard PCR Assay - Tg(TcrHEL3A9)1Mmd
Version 2.2
Notes
Primers amplify from the transgene and also amplify a fragment from the endogenous Tcr locus.
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
TG = 371 bp
IPC = 415 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 |
|---|---|---|---|---|---|---|
| 21238 | CTG TCC CTG TAT GCC TCT GG | Internal Positive Control Forward | A | |||
| 21239 | AGA TGG AGA AAG GAC TAG GCT ACA | Internal Positive Control Reverse | A | |||
| oIMR0572 | GCA GTC ACC CAA AGC CCA AG | Transgene Forward | A | Tcrb | ||
| oIMR0573 | CCC CAG CTC ACC TAA CAC TG | Transgene Reverse | A | Tcrb |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21238 | 0.50 uM |
| 21239 | 0.50 uM |
| oIMR0572 | 0.50 uM |
| oIMR0573 | 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.