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Notes
Taqman qPCR protocols are run on a real time PCR instrument. 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
Tg= 114 bp
IPC = 74 bp
X-linked
For Stock No. 010905- This assay is used to detect the spontaneous translocation [T(XTmsb4x-Hccs;Y)1Dto] to the YSry- chromosome. This is a 3.2 MB region mapping to Chromosome X which contains nine annotated genes (Tmsb4x, Tlr8, Tlr7, Prps2, Frmpd4, Msl3, Arhgap6, Amelx and Hccs) . This assay is specifcally used to determine copy number of Tlr7.
Stock No. 039108 contains the YSry- chromosome without the 3.2 MB translocation.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 73409 | CCC TTA CCA TCA ACC ACA TAC C | Forward | A | |||
| 73410 | CTT TGG ACC CCA GTA GAA CAG | Reverse | A | |||
| 73411 | Fluorophore-1 | TTC CGT AGG CTG AAC CAT CTG GAA G | 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 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa Probe Fast QPCR | 1.00 X |
| 73409 | 0.40 uM |
| 73410 | 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.