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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:99033984+99034082 99bp TTGAAGTATAGTCCTAGGCAGGAG GGGAAGAACGTGTGAGAATG
Mut= 98 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Mut Sequence:
ttgaagtatagtcctaggcaggagtggccatgatatgacagccatcactgtccctaactgaatcatggattcatgactcattgagtgtgcccagttga
This mutation is a 4195 bp deletion beginning at Chromosome 15 position 99,034,012 bp and ending after 99,038,208 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48219 | TTG AAG TAT AGT CCT AGG CAG GAG | Common | A | |||
48220 | GGG AAG AAC GTG TGA GAA TG | Wild type Reverse | A | |||
48221 | TCA ACT GGG CAC ACT CAA TG | Mutant Reverse | A | |||
48222 | Fluorophore-1 | CCA TGC CCC AAG GTC TC | Quencher-1 | WT Probe | ||
48223 | Fluorophore-2 | ACA GCC ATC ACT GTC CCT AAC T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48219 | 0.40 uM |
48220 | 0.40 uM |
48221 | 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 |