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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.
primer tmoimr0043 changed to 14241. 3/5/12
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14241 | Fluorophore-1 | TGG TCT CGT CCA TCA GCG CCC T | 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 | |||
oIMR6512 | GCT CCC AGG TCA CCC AGG AA | Transgene Forward | A | |||
oIMR7432 | TCC GAT TTG TAG GCC TTC AAC T | Transgene Reverse | A | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa Probe Fast QPCR | 1.00 X |
oIMR1544 | 0.40 uM |
oIMR3580 | 0.40 uM |
oIMR6512 | 0.40 uM |
oIMR7432 | 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 |