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Stock No: 012478
Protocol 20090: QPCR Assay - Tg(HLA-DRA)-qPCR
Version 2.0
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
Sequence
CATGATTTCCAGGTTGGCTTTGTCCACAGCTATGTTGGCCAATGCACCTTGAGCCTCAAAGCTGGCAAATCGTCCAAATTCTT
CAAGCCGCCAGACCGTCTCCTTCTTTGCCATATCCACATGGAAAATCTCATCACCATCAAAGTCAAACATAAACTCGCCTGAT
TGGTCAGGATTCAGATAGAACTCGGCCTGGATGATCACATGTTCTTCTGAAAGGCAAGAAATGGAGAAAGAGAGTTGGGTG
GGGGGAATCA
CAAGCCGCCAGACCGTCTCCTTCTTTGCCATATCCACATGGAAAATCTCATCACCATCAAAGTCAAACATAAACTCGCCTGAT
TGGTCAGGATTCAGATAGAACTCGGCCTGGATGATCACATGTTCTTCTGAAAGGCAAGAAATGGAGAAAGAGAGTTGGGTG
GGGGGAATCA
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 12023 | GCT ATG TTG GCC AAT GCA CCT TGA | Transgene Forward | A | HLA-DRA | ||
| 12024 | AGA ACA TGT GAT CAT CCA GGC CGA | Transgene Reverse | A | HLA-DRA | ||
| 12025 | Fluorophore-1 | AAA TTC TTC AAG CCG CCA GAC CGT CT | Quencher-1 | |||
| oIMR1544 | CAC GTG GGC TCC AGC ATT | Internal Positive Control Forward | A | |||
| oIMR3580 | TCA CCA GTC ATT TCT GCC TTT G | Internal Positive Control Reverse | A | |||
| TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa Probe Fast QPCR | 1.00 X |
| 12023 | 0.40 uM |
| 12024 | 0.40 uM |
| oIMR1544 | 0.40 uM |
| oIMR3580 | 0.40 uM |
| Tg Probe | 0.15 uM |
| IC Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 035855 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)Dvs Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/J |
| 017914 | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ |
| 030331 | NOD.Cg-Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl/SzJ |
| 012479 | NOD.Cg-Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Prkdcscid Il2rgtm1Wjl/GckRolyJ |
| 012478 | NOD.Cg-Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Prkdcscid/GckJ |
| 026566 | STOCK Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz Tg(TcraTcrb)1Lkm/J |
| 6 strains use this protocol | |