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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.
Mutant= 134 bp
Wild Type = 139 bp
>chr10:93216463+93216601 139bp GGATCTCAATGGAGGTAACCA GGCAAAGAAACAGCTCTTGTG
Wt Sequence:
Mutant Sequence:
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36717 | TGT CAG GTT GAC CCA AAA CA | Mutant Forward | A | |||
36718 | GGC AAA GAA ACA GCT CTT GTG | Common | A | |||
36719 | GGA TCT CAA TGG AGG TAA CCA | Wild type Forward | A | |||
36720 | Fluorophore-1 | AGC ACA GCT GGC CTG AGA | Quencher-1 | MUT Probe | ||
36721 | Fluorophore-2 | AGT GGA AAC GCA ACC ATT GT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36717 | 0.40 uM |
36718 | 0.40 uM |
36719 | 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 |