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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:18174296-18174415 120bp GCTAGAGTTAAAGTCGCAGAATCCTA TGAGAAGTGGTTTTGGTGACTA
Mutant= 118 bp
Wild Type = 120 bp
The wildtype reverse primer (primer 65472) anneals over the nucleotide sequence containing mouse genomic variation rs33609550.
Wt Sequence: GCTAGAGTTAAAGTCGCAGAATCCTAtaagaatgctgggtaagtgtctcaaggtggcatatacacaaatacatataaacttatataggaggtaatataTAGTCACCAAAACCACTTCTCA
Mut Sequence: gctagagttaaagtcgcagaatcctataagaatgctgggtaagtgTTGAACcttttcctggtacgcagatgctgaacaatggatttacaaacagatacccatataggagatgatgctg
A 499 bp deletion beginning at Chromosome 10 position 18,173,871 bp and ending after 18,174,369 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65471 | GCT AGA GTT AAA GTC GCA GAA TCC TA | Common | A | |||
65472 | TGA GAA GTG GTT TTG GTG ACT A | Wild type Reverse | A | |||
65473 | CAG CAT CAT CTC CTA TAT GGG TAT C | Mutant Reverse | A | |||
65474 | Fluorophore-1 | TTG AAC CTT TTC CTG GTA CGC | Quencher-1 | MUT Probe | ||
65475 | Fluorophore-2 | CTC AAG GTG GCA TAT ACA CAA ATA CA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65471 | 0.40 uM |
65472 | 0.40 uM |
65473 | 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 |