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Primers in this protocol are not specific for the transgene. WT samples will have PCR product but will have a lower copy number.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
10102 | Fluorophore-1 | CAG CAG ATT CTC AGT CCA ACA G | Quencher-1 | Transgene Forward | A | |
10103 | Fluorophore-2 | GCA CAG AAG TAC ACG GCA GA | Quencher-2 | Transgene Reverse | A | |
13646 | Fluorophore-3 | CTC TAG CTC CAA GGC ACT CAT GTT CAT CTC A | Quencher-3 | 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-4 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-4 | IC Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa Probe Fast QPCR | 1.00 X |
10102 | 0.40 uM |
10103 | 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 |