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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut=114 bp
Wt=114 bp
Wt Sequence (bp changes in brackets with wt first):
ATTGCGACCTCTGTGGTAGGCCATTTCCAGCCTGCCCAAGTAACCAGAGCCCCTTACCCCTCAGATCA(cc/tg)CTGACGACCATTGGCTACGGGGACAAGTACCCTCAGACCTGGAA
Kcnq2 T274M (ACC -> ATG)
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39097 | ATT GCG ACC TCT GTG GTA GG | Forward | A | |||
39098 | TTC CAG GTC TGA GGG TAC TTG | Reverse | A | |||
39099 | Fluorophore-1 | TAC CCC TCA GAT CAC CCT GA | Quencher-1 | WT Probe | ||
39100 | Fluorophore-2 | CCT TAC CCC TCA GAT CAT GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39097 | 0.40 uM |
39098 | 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 |