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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
TG (DQB1) = 260 bp
internal control (DQB1) = 200 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 |
|---|---|---|---|---|---|---|
| oIMR8692 | AGG ATT TCG TGT ACC AGT TTG AGG GCA T | Transgene Forward | A | |||
| oIMR8693 | TGC AAG GTC GTG CGG AGC TCC AA | Transgene Reverse | A | |||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | A | |||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR8692 | 0.50 uM |
| oIMR8693 | 0.50 uM |
| oIMR8744 | 0.50 uM |
| oIMR8745 | 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
| Stock Number | Strain Name |
|---|---|
| 039054 | NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-A/H2-D/B2M)1Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ |
| 039056 | NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv Tg(CD2-TcraA1.9,Cd4-TcrbA1.9)1Dvs Tg(INS*)172Dvs/DvsJ |
| 039053 | NOD.Cg-H2-K1d-em1Dvs H2-Ab1g7-em1Dvs H2-D1b-em5Dvs Tg(HLA-DQA1,HLA-DQB1)1Dv/DvsJ |
| 004606 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ |
| 026561 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ |
| 5 strains use this protocol | |