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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr16:38415643-38415752 110bp TGGCTGGAATCTGCTATGTG CCAGATGTGGAACCAGCAC
Mutant= 83 bp
Wild Type = 110 bp
Wt Sequence: tggctggaatctgctatgtgttgtgagttgtacatgcaAcgagaggccctttcagataatttacatgtgcggatgctggaaagctccacgtgtgctggttccacatctgg
Mut Sequence: tggctggaatctgctatgtgttgtgagttgtacatgcaAGtcacaagacatggtattgcacaaaaataaggaatgctgactgt
A 544 bp deletion beginning at Chromosome 16 position 38,415,170 bp and ending after 38,415,713 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65536 | TGG CTG GAA TCT GCT ATG TG | Common | A | |||
65537 | ACA GTC AGC ATT CCT TAT TTT TGT G | Mutant Reverse | A | |||
65538 | CCA GAT GTG GAA CCA GCA C | Wild type Reverse | A | |||
65539 | Fluorophore-1 | TAC ATG CAA GTC ACA AGA CAT GGT | Quencher-1 | MUT Probe | ||
65540 | Fluorophore-2 | AGG CCC TTT CAG ATA ATT TAC ATG T | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65536 | 0.40 uM |
65537 | 0.40 uM |
65538 | 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 |