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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mut= 112 bp
Wt= 112 bp
Wt Sequence:AGCATTATGCCAACATCTGGCAGGGCAAGTTTCCTGTGAGCAACACTGGCGAGGATGGCTTCCAAGGAACTGccCCCGTGAGTAAGCAGGCTTCTTGAGAAGGGCTGTGTGC
Mutant Sequence:AGCATTATGCCAACATCTGGCAGGGCAAGTTTCCTGTGAGCAACACTGGCGAGGATGGCTTCCAAGGAACTGtaCCCGTGAGTAAGCAGGCTTCTTGAGAAGGGCTGTGTGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38585 | AGC ATT ATG CCA ACA TCT GG | Forward | A | |||
38586 | GCA CAC AGC CCT TCT CAA G | Reverse | A | |||
38587 | Fluorophore-1 | AAC TGC CCC CGT GAG TAA G | Quencher-1 | WT Probe | ||
38588 | Fluorophore-2 | CCA AGG AAC TGT ACC CGT GA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38585 | 0.40 uM |
38586 | 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 |