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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:38574214+38574314 101bp GCTGTGTAAACCCCAGAGC AGGTCCGGCTTAAAAACGAG
Mutant= 99 bp
Wild Type = 101 bp
Wt Sequence: gctgtgtaaaccccagagctccgtgccccggctccgattacAaccatgtgtcTggtgttgattgcagGAGGCCAGAGCAAGCTCGTTTTTAAGCCGGACCT
Mut Sequence: gctgtgtaaaccccagagctccgtgccccgatatatatatatgtatacacacacacacacacacatatatatgtatatatgcatttgcctgcacgtatg
This mutation is a 807 bp deletion beginning at Chromosome 16 position 38,753,882 bp and ending after 38,754,688 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
64984 | AGG TCC GGC TTA AAA ACG AG | Wild type Reverse | A | |||
64985 | ATT CAC TTG CAT ACG TGC AG | Mutant Reverse | A | |||
65943 | Fluorophore-1 | TCT GGT GTT GAT TGC AGG AG | Quencher-1 | WT Probe | ||
66231 | Fluorophore-2 | CCG TGC CCC GAT ATA TAT ATA TGT ATA C | Quencher-2 | MUT Probe | ||
66232 | GCT GTG TAA ACC CCA GAG C | Common | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
64984 | 0.40 uM |
64985 | 0.40 uM |
66232 | 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 |