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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.
3' end has lots of gt repeats - had to make large products and mut probe is across a string of 5g's which im not thrilled about.
Mutant= 96 bp
Wild Type =138 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
31973 | CCA CAT CCT ACC CTT TCC AC | Common | A | |||
31975 | Fluorophore-1 | CCC TGA GGG GGT GAA TAA AT | Quencher-1 | WT Probe | ||
31980 | CTC CCC ACT GAA CAG ACA CA | Wild type Reverse | A | |||
33697 | Fluorophore-2 | ACT GTC AGA GAC GGG GGT G | Quencher-2 | MUT Probe | ||
33698 | CCA AGC AGT TAG CAC CAC AC | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
31973 | 0.40 uM |
31980 | 0.40 uM |
33698 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |