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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.
>chr19:34312593+34312684 92bp GTGCCCTAGGAAACACAGCA CTCTAGCCAGGAATACAAATGACA
Mutant= 110 bp
Wild Type = 92 bp
Wt Sequence: gtgccctaggaaacacagcatagattccatttgctgctgtgtccatgtGGagcagatttgactgcgtttgtcatttgtattcctggctagag
Mutant Sequence: gtgccctaggaaacacagcatagattccatttgctgctgtgtccatgtgTGTAGTCTCCCCTCCCCCAGCCTGAAACCTGCTTGCTCGGGGTGGAGCTTCCTGCTCATTC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
35342 | Fluorophore-1 | TGT GTG TAG TCT CCC CTC CC | Quencher-1 | MUT Probe | ||
35343 | Fluorophore-2 | ATG TGG AGC AGA TTT GAC TGC | Quencher-2 | WT Probe | ||
35344 | GTG CCC TAG GAA ACA CAG CA | Common | A | |||
35345 | GAA TGA GCA GGA AGC TCC AC | Mutant Reverse | A | |||
35346 | CTC TAG CCA GGA ATA CAA ATG ACA | Wild type Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
35344 | 0.40 uM |
35345 | 0.40 uM |
35346 | 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 |