Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 82 bp
Wild Type = 115 bp
>chr12:86817366+86817480 115bp CTCCTCCGACTCCCTGAG TCTCCTCTCTGTAATGCACCAG
Wt Sequence: AAAGGGCAGCAGCAGCAACGAGCCCTCCTCCGACTCCCTGAGCTCACCCACGCTGCTGGCCCtgtgAGCAGTCAGAGAAGGCAAGGCAGCCGGCATCCAGACGTGCCACTGCCCGAGCTGGTGCATTACAGAGAGGAGAAACACGTCTTCCCTCGAAGGTTCCCGTCGACCTAGGGAGGACCTTACCTGTTCGTGAAACACACCAGGCTGTGGGCCTCAAGGACTTGCAAgcaTCCACATCTGGCCTCCAGTCCTCACCTCTTCCAGAGA
Mutant Sequence:GAGCCCTCCTCCGACTCCCTGAGCTCACCCACGCTGCTGGCCCTGggtagtggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgccagagccagcgaagtctgctcccgcccCGAAAAAGGGCTCCAAGAAGGCGGTGACTAAGGCGCAGAAGAAAGACGGCAAGAAGCGCAAGCGCAGCCGCAAGGAGAGCTATTCCATCTATGTGTACAAGGTTCTGAAGCAGGTCCACCCTGACACCGGCATTTCGTCCAAGGCCATGGGCATCATGAATTCGTTTGTGAACGACATTTTCGAGCGCATCGCAGGTGAGGCTTCCCGCCTGGCGCATTACAACAAGCGCTCGACCATCACCTCCAGGGAGATCCAGACGGCCGTGCGCCTGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39764 | Fluorophore-1 | CTC CTC CGA CTC CCT GAG | Quencher-1 | Common | A | |
39765 | GTC ACC GCA TGT TAG CAG AC | Mutant Reverse | A | |||
39766 | Fluorophore-2 | TCT CCT CTC TGT AAT GCA CCA G | Quencher-2 | Wild type Reverse | A | |
39767 | Fluorophore-3 | CCC TGG GTA GTG GAG AGG GC | Quencher-3 | MUT Probe | ||
39768 | Fluorophore-4 | CCC TGT GAG CAG TCA GAG AAG GC | Quencher-4 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39764 | 0.40 uM |
39765 | 0.40 uM |
39766 | 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 |