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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:9028106-9028245 140bp GGGAGCTGTCCACACTGTTA GTTTGACATTTCCTGTAAAGATTGT
Mutant= 114 bp
Wild Type = 140 bp
Wt Sequence: gggagctgtccacactgttaaaattctgcagattagggaattcaggtccatgtccatgaaaCagaaggtaaatgttcaagcagttggcaaagattaataacaaaaaactatttttacaatctttacagGAAATGTCAAAC
Mut Sequence: gggagctgtccacactgttaaaattctgcagattagggaattcaggtccatgtccatgaaaCCgtaggagacatacaccactgtatattattatgaaccacaatgacactcctt
A 409 bp deletion beginning at Chromosome 11 position 9,027,775 bp and ending after 9,028,183 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65546 | GGG AGC TGT CCA CAC TGT TA | Common | A | |||
65547 | AAG GAG TGT CAT TGT GGT TCA T | Mutant Reverse | A | |||
65548 | GTT TGA CAT TTC CTG TAA AGA TTG T | Wild type Reverse | A | |||
65549 | Fluorophore-1 | AGG TAA ATG TTC AAG CAG TTG GC | Quencher-1 | WT Probe | ||
65550 | Fluorophore-2 | CCA TGA AAC CGT AGG AGA CAT ACA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65546 | 0.40 uM |
65547 | 0.40 uM |
65548 | 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 |