Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 80 bp
Wild Type = 86 bp
>chr14:54545657-54545742 86bp CCATGACCCCAGTTCAGGT CACATCGAACTGAAAAAGCTG
CCAAATGTTAGGATTAGTGTACCACCATTCCTGGCTCTCGTGATGCCAGTTGTTTGGTTATGAGGCTCTCTGTGTTCTCACACCACTGGCAGGTCTCCCTCGTGctaacagtctgtgtgcctctaggatttcgtgtggatgaaagcccggctccagctggaggtggaggagcagctcaagaggaaatgcttcacgctgctgtgctaccatgaccccagttcaggtgtgagcctccccggccTGCTCATGTGCCCTCAGTTCCTGGGCTAGCCAGCTTTTTCAGTTCGATGTGGGTGTGAGACAGGGTACTCTGTATTCCAGGGTGTCTAGCCAGGAACTCACTATGTTACCCAAGTTGGTCTCAAACCTGTGGCAAGCCTGCCTGGGTGCTCCAAGTGTTGAGATTATTGGCATGACCCACCATGCCAACTTTAGACTACTCTGAGACTTTAGAATCAGAAATGATGGTTTATTTCCTCAGGAGTGGGTTT
Mutation details
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44194 | CCA TGA CCC CAG TTC AGG T | Wild type Forward | A | |||
44195 | CAC ATC GAA CTG AAA AAG CTG | Common | A | |||
44196 | TCT CAC ACC ACT GGC AGG T | Mutant Forward | A | |||
44197 | Fluorophore-1 | CCC GGC CTG CTC ATG TG | Quencher-1 | WT Probe | ||
44198 | Fluorophore-2 | CCC TCG TGT GCT CAT GTG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44194 | 0.40 uM |
44195 | 0.40 uM |
44196 | 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 |