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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.
>chr2:156921239+156921333 95bp CACACTCAACCAGCCCATC ACTCTGGGACTTCCCCATCT
Mutant= 92 bp
Wild Type = 95 bp
Wt Sequence: cacactcaaccagcccatccccatcacacgcttagtgacatcttagtgtgacgtgactgggaacgaGGagtcacaagatggggaagtcccagagt
Mutant Sequence: ttgaaagcaggaagctttggaggtttgattgttagcatttatttggcactgccaggatgcaggGGagtcacaagatggggaagtcccagagt
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34543 | CAC ACT CAA CCA GCC CAT C | Wild type Forward | A | |||
34544 | ACT CTG GGA CTT CCC CAT CT | Common | A | |||
34545 | TTG AAA GCA GGA AGC TTT GG | Mutant Forward | A | |||
34546 | Fluorophore-1 | CAC ACG CTT AGT GAC ATC TTA GTG | Quencher-1 | |||
34547 | Fluorophore-2 | TGG CAC TGC CAG GAT GC | Quencher-2 |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
34543 | 0.40 uM |
34544 | 0.40 uM |
34545 | 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 |