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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:51491479+51491581 103bp TCGCTGCGTATACATCTGTG ACTTCAGTGTCCCAAGGATTTAC
Mut = 122 bp
Wt = 103 bp
Fam = Mut
Hex = Wt
Wt Sequence:
Mutant Sequence:
This mutation is a 137 bp deletion beginning at Chromosome 10 position 51,481,213 bp and ending after 51,481,349 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52948 | TCG CTG CGT ATA CAT CTG TG | Common | A | |||
52949 | ACT TCA GTG TCC CAA GGA TTT AC | Wild type Reverse | A | |||
52952 | Fluorophore-1 | AGA GTG CTG CTG GCT TCT CA | Quencher-1 | MUT Probe | ||
54744 | TTC CCT GAT GTC CTC TCA CC | Mutant Reverse | A | |||
55122 | Fluorophore-2 | ATC TGG TGT TGG GGT TCC TG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52948 | 0.40 uM |
52949 | 0.40 uM |
54744 | 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 |