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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.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
20454 | GAG GCT GAA GCT GAA GGA C | Transgene Forward | A | |||
20455 | GAT GGT GTA GTC CTC GTT GTG | Transgene Reverse | A | |||
20456 | Fluorophore-1 | CCA ACT TGA TGT TGA CGT TGT AGG CG | 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 | |||
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 |
20454 | 0.40 uM |
20455 | 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 |
---|---|
021577 | B6;D2-Tg(Myh6*-mCherry)2Mik/J |
024046 | C57BL/6-Tg(Csf1r-HBEGF/mCherry)1Mnz/J |
018542 | FVB-Tg(Kdr-mCherry)1Medi/J |
034270 | NOD/ShiLtJ-Flt3em1Doi/J |
027967 | STOCK Tg(CAG-Arl13b/mCherry)1Kvand Tg(CAG-EGFP/CETN2)3-4Jgg/KvandJ |
025693 | STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ |
6 strains use this protocol |