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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.
Mouse wt = 80 bp
IPC = 74 bp
Plese note this is a qPCR for mouse Exon 14 on the X chromosome so Female mouse wt will have two copies (hom-like); Female hets and wt Males will have one copy (tg/o-like) and Female homs and Male hemis will have no copies (wt-like)
X-linked
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33653 | Fluorophore-1 | CAA CGT ACT GCA AAC CCG CAA GTA TT | Quencher-1 | |||
34225 | GAT CAA ATT TTC AGA ATC GGA CTA C | Transgene Forward | A | |||
34226 | GCC AGA AGA AAT CAG ACT GTG C | Transgene Reverse | 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 | |||
TmoIMR0105 | Fluorophore-2 | CCA ATG GTC GGG CAC TGC TCA A | Quencher-2 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa Probe Fast QPCR | 1.00 X |
34225 | 0.40 uM |
34226 | 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 |