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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:88689733+88689833 101bp GAAGTCTTTAAGGTTGCCAGGA TCAATAGGTCATGTTAGACCAAGC
Mutant= 102 bp
Wild Type = 101 bp
Wt Sequence: gaagtctttaaggttgccaggaaatgtatatagttcaatactttccCctcagatggagccagtaagtttagaatttagcttggtctaacatgacctattga
Mut Sequence: gaagtctttaaggttgccaggaaatgtatatagttcaatactttccCCtcagtgcagctatgcatcagagtgaggacattcctgacactgtagcatgtggga
A 406 bp deletion beginning at Chromosome 17 position 88,689,780 bp and ending after 88,690,185 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65482 | GAA GTC TTT AAG GTT GCC AGG A | Common | A | |||
65483 | TCA ATA GGT CAT GTT AGA CCA AGC | Wild type Reverse | A | |||
65484 | TCC CAC ATG CTA CAG TGT CA | Mutant Reverse | A | |||
65485 | Fluorophore-1 | TAC TTT CCC CTC AGT GCA GCT A | Quencher-1 | MUT Probe | ||
65486 | Fluorophore-2 | CCT CAG ATG GAG CCA GTA AGT T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65482 | 0.40 uM |
65483 | 0.40 uM |
65484 | 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 |