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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:44102579+44102694 116bp ACTACCACCCCAGCAAGGAC CTTTCCTCCCCAACACCATC
Mutant= 112 bp
Wild Type = 116 bp
Wt Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTaagtCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG
Mutant Sequence: CACTACCACCCCAGCAAGGACAGGGCAGCAGGAGGCAGCCATGAGCCCTACACACCAGACCAGTCCCAAGGGCACTCTCTGGAGAAGGCCCAGGATGGTGTTGGGGAGGAAAG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38489 | ACT ACC ACC CCA GCA AGG AC | Forward | A | |||
38490 | CTT TCC TCC CCA ACA CCA TC | Reverse | A | |||
38491 | Fluorophore-1 | CAG ACC AGT AAG TCC CAA GGG | Quencher-1 | WT Probe | ||
38492 | Fluorophore-2 | CAG ACC AGT CCC AAG GGC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38489 | 0.40 uM |
38490 | 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 |