Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 119 bp
Wild Type = 118 bp
>chr3:107556725-107556842 118bp CCTCCTGGTTCCTGTATTCC GTTAGCATTGGGGGTGTTGT
gctaacctcctggttcctgtattccagGATGATCCTGTTTGCTACCTAAagaaggcctttttt(-/T)ctggtacaagacataATAGATGAGACCATGCGCTTTAAAGACAACACCCCCAATGCTAACGCCACCGAGAGGCTCCAGGAACTCTC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39024 | CCT CCT GGT TCC TGT ATT CC | Forward | A | |||
39025 | Fluorophore-1 | AGG CCT TTT TTT CTG GTA CAA GAC AT | Quencher-1 | MUT Probe | ||
39026 | Fluorophore-2 | AGG CCT TTT TTC TGG TAC AAG ACA T | Quencher-2 | WT Probe | ||
9998 | Fluorophore-3 | GTT AGC ATT GGG GGT GTT GT | Quencher-3 | Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39024 | 0.40 uM |
9998 | 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 | 4.0 | -- | Forever |