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Stock No: 006024
Protocol 24041: Separated PCR Assay - H2-Ab1<b-tm1Gru>alternate2
Version 1.2
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
Mutant = ~295 bp
Heterozygote = 185 bp and ~295 bp
Wild type = 185 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 15442 | ATG GGC GAG TGC TAC TTC AC | Wild type Forward | B | |||
| 15801 | GCC CGC GTT CGC TCC AGG | Common | A, B | |||
| oIMR1100 | GCT ATC AGG ACA TAG CGT TGG | Mutant Forward | A | neo F |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 15801 | 0.50 uM |
| oIMR1100 | 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.
Reaction B
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTPS-kapa | 0.26 mM |
| 15442 | 0.50 uM |
| 15801 | 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 |
|---|---|
| 038341 | B6.Cg-H2-Ab1b-tm1Gru Tg(Itgax-H2-Ab1)1Tml/J |
| 038339 | B6.Cg-H2-Ab1b-tm1Gru Tg(KRT14-H2-Ab1)1Glm/TmlJ |
| 002956 | B6NTac.Cg-H2-Ab1b-tm1Gru Tg(Ab1TL)1Gru/J |
| 004256 | NOD.129S2(B6)-H2-Ab1b-tm1Doi/DvsJ |
| 006021 | NOD.Cg-H2-Ab1b-tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ |
| 006023 | NOD.Cg-H2-Ab1b-tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ |
| 026561 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ |
| 021885 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl/SzJ |
| 006022 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ |
| 006024 | NOD.Cg-Rag1tm1Mom H2-Ab1b-tm1Gru Tg(CD4,HLA-DQA1,HLA-DQB1)N8Ell/EllJ |
| 030331 | NOD.Cg-Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl/SzJ |
| 11 strains use this protocol | |