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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:53011566+53011668 103bp GACCCACTCTTCTGGGTTCC GTATTCTGCATCCCCATTGC
Mutant= 76 bp
Wild Type = 103 bp
Wt Sequence: gacccactcttctgggttccaggaaaattatccCatctttgttcggagactcccaggccatgctggggatggggtttggagttgcaatggggatgcagaatac
Mut Sequence: gacccactcttctgggttccaggaaaattatccCGtggaatagcatgcttgctggctctgagggcgagcttctgta
A 10,576 bp deletion beginning at Chromosome 4 position 53,011,600 bp and ending after 53,022,175 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65541 | GAC CCA CTC TTC TGG GTT CC | Common | A | |||
65542 | GTA TTC TGC ATC CCC ATT GC | Wild type Reverse | A | |||
65545 | Fluorophore-1 | TCT TTG TTC GGA GAC TCC CA | Quencher-1 | WT Probe | ||
66498 | TAC AGA AGC TCG CCC TCA GA | Mutant Reverse | A | |||
66499 | Fluorophore-2 | TTA TCC CGT GGA ATA GCA TGC TT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65541 | 0.40 uM |
65542 | 0.40 uM |
66498 | 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 |