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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.
Oligo 14576 replaces TmoIMR0082
The Target probe is for WILD TYPE. Therefore, Homs do NOT amplify for target.
Wild Type will appear below hets on the graph.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14576 | Fluorophore-1 | CAT CCG TCT TGC ATA ATG CTT GCT GTG A | Quencher-1 | Tg Probe | A | |
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 | |||
oIMR6166 | GTG GCT GGG TTG CTT CTC A | Transgene Forward | A | |||
oIMR6167 | CCC TCA ATG TTC CGT GCT G | Transgene Reverse | A | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 | IC Probe | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa Probe Fast QPCR | 1.00 X |
14576 | 0.40 uM |
oIMR1544 | 0.40 uM |
oIMR3580 | 0.40 uM |
oIMR6166 | 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 |