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.
Mutant= 111 bp
Wild Type = 144 bp
Wt Sequence:tggataccatggagagagtccagcagcaaccaagggtgggttgctaccaccactgcgtgctatcctgaccactatgaatagtcacatgcccagctgctggcCCacagaggctggagacagatctgccctgatgtggttcctggctccagggctagcatgtgctccacagcggaaatgagactcctcaagggaaggggtggggggt
Mutant Sequence:agacagcattggataccatggagagagtccagcagcaaccaagggtgggttgctaccaccactgcgtgctatcCTGACCACTATGAATAGTCACATGCCCAGCTGCTGGCCaattgtatcacgcgtataacttcgtatagcatacattatacgaagttatgccactagaggatccccgCACAGAGGCTGGAGACAGATCTGCCCTGATGTGGTTCCTGGCTCCAGGGCTAGCATGTGCTCCACAGCGGaaatgagactcctcaagggaaggggtgggg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
34728 | Fluorophore-1 | AGT TAT GCC ACT AGA GGA TCC C | Quencher-1 | MUT Probe | ||
34729 | Fluorophore-2 | TGC TGG CCC ACA GAG G | Quencher-2 | WT Probe | ||
35055 | GCT GGC CAA TTG TAT CAC G | Mutant Forward | A | |||
35056 | AGC ATT GGA TAC CAT GGA GAG | Wild type Forward | A | |||
35057 | GAA CCA CAT CAG GGC AGA | Common | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35055 | 0.40 uM |
35056 | 0.40 uM |
35057 | 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 |