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Stock No: 016133
Protocol 8743: Standard PCR Assay - Tg(Defa2-Myd88)1Lvh
Version 2.0
Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
This assay does not work well without the use of a Hotstart 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
Transgene = ~350 bp
Positive Internal Control = 200 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12093 | AGG TGG CCT TTG ACA CCT ACC AGG | Transgene Forward | ||||
| 12094 | TCT GTT GTG TTT CCT CCC TGT TGG | Transgene Reverse | ||||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control | ||||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.00 |
| MgCl2 | 2.00 |
| dNTP KAPA | 0.20 |
| oIMR8744 | 0.50 |
| oIMR8745 | 0.50 |
| 12093 | 0.50 |
| 12094 | 0.50 |
| Kapa 2G HS taq polym | 0.01 |
| EvaGreen | 1.00 |
| DNA | 0.00 |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -1.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles | |
| 6 | 94.0 | -- | |
| 7 | 50.0 | -- | |
| 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.
Strains Using This Protocol
This is the only strain that uses this protocol.