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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.
Tg= 145 bp
IPC = 74 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
25140 | GCT GAA CTA ACC TAT TTA TGT TGG ATG | Transgene Forward | A | |||
25141 | GCG TCT TGT ATT TAA ACT GGA GTG | Transgene Reverse | A | |||
25142 | Fluorophore-1 | TGG CTC TCT TGA TTG CTG TTC CGT | 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 |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
25140 | 0.40 uM |
25141 | 0.40 uM |
oIMR1544 | 0.40 uM |
oIMR3580 | 0.40 uM |
Tg Probe | 0.15 uM |
IC Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | repeat steps 2-3 for 40 cycles |
Stock Number | Strain Name |
---|---|
034712 | B6.Cg-Cyp27a1tm1.1Dpm/J |
005703 | B6.Cg-Tg(ACTFLPe)9205Dym/J |
003800 | B6;SJL-Tg(ACTFLPe)9205Dym/J |
034731 | B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J |
019100 | B6N.Cg-Tg(ACTFLPe)9205Dym/CjDswJ |
035185 | STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ |
6 strains use this protocol |