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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:24422533+24422630 98bp GCCATCACACTTCATCAGCA CTTCCAGTTGTGCTTTGTGC
Mutant= 115 bp
Wild Type = 98 bp
Wt Sequence: gccatcacacttcatcagcactaatttttttcactgtgtagtacaactgactctcatttccccccagGTCCGTTAAATGCACAAAGCACAACTggaag
Mutant Sequence: gccatcacacttcatcagcactaatttttttcactgtgtagtacaactgactctcatttccccccaGGAAGTTAGACGTGAGCACCATGAAATATATCAACCCTCCAGGGAGCTT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14414 | CTT CCA GTT GTG CTT TGT GC | Wild type Reverse | A | |||
43144 | GCC ATC ACA CTT CAT CAG CA | Common | A | |||
43145 | AAG CTC CCT GGA GGG TTG A | Mutant Reverse | A | |||
43146 | Fluorophore-1 | TTT CCC CCC AGG TCC GT | Quencher-1 | WT Probe | ||
43147 | Fluorophore-2 | CCC CCA GGA AGT TAG ACG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
14414 | 0.40 uM |
43144 | 0.40 uM |
43145 | 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 |