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.
>chr8:88402130+88402304 175bp CTTGGCGCTAGCATACAGG CCACACTTAAAAGAGTTGATGGT
Mutant= 160 bp
Wild Type = 175 bp
Wt Sequence: CTTGGCGCTAGCATACAGgtgggctgccgtctgttctcctgctaactccacgtggacgAGttttatagaattagtgctttaaaagaacactacaaagtatattttggagatcatttgatatatttgagtatagactgtatattacataacataccatcaactcttttaagtgtgg
Mutant Sequence: ggaggctttcttcgcatgtataaagacaactttgtgcagtatgGGttttatagaattagtgctttaaaagaacactacaaagtatattttggagatcatttgatatatttgagtatagactgtatattacataacataccatcaactcttttaagtgtgg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
33950 | Fluorophore-1 | AGA CAA CTT TGT GCA GTA TGG GT | Quencher-1 | MUT Probe | ||
33951 | Fluorophore-2 | TGC CGT CTG TTC TCC TGC | Quencher-2 | WT Probe | ||
33952 | CTT GGC GCT AGC ATA CAG G | Wild type Forward | A | |||
33953 | CCA CAC TTA AAA GAG TTG ATG GT | Common | A | |||
33954 | GGA GGC TTT CTT CGC ATG T | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
33952 | 0.40 uM |
33953 | 0.40 uM |
33954 | 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 |