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Stock No: 006021
Protocol 22118: QPCR Assay - DQ8 Beta
Version 3.0
Notes
Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. Use an appropriate instrument-specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing δCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
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
26561 can be used as controls for 39053, 39054, 39055, 39056, & 36581. LM 3-6-24
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9143 | GTG GAG GCA AAA TCT TGG TAC TG | Transgene Forward | A | |||
| 9144 | CAC CAT TTG CCC ATA AAT CAG | Transgene Reverse | A | |||
| 9145 | Fluorophore-1 | CTG GAT GAC CAC AGA CAG GGA GTC TTC | Quencher-1 | Tg Probe | ||
| 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 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 9143 | 0.40 uM |
| 9144 | 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 |
|---|---|
| 006021 | NOD.Cg-H2-Ab1b-tm1Gru Tg(CD2-CD4,HLA-DQA1,HLA-DQB1)1Ell/EllJ |
| 004606 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ |
| 026936 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv Tg(INS*)172Dvs/DvsJ |
| 026561 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl Tg(HLA-DQA1,HLA-DQB1)1Dv/SzJ |
| 4 strains use this protocol | |