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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 88 bp
Wild Type = 102 bp
Wt Sequence: tgagaagatcccgatacctacaaagatttgtcctcctgcaggatcgtgggatcctgcccagcggtcctatctagaggtcattctctccacagAGCGAGTCAAGAACCACTGGCAGGAAGACCTCATGTTTGGCTACCAGTTCCTGAATGGCTGCAACCCAGTACTCATCAAGCGtTGCACAGCGTTGCCCCCGAAGCTCCCAGTGACCACAGAGATG
Mutant Sequence: gcaaaaccaaattaagggccagctcattcctcccactcatgatctatagatctatagatctctcgtgggatcattgtttttctcttgattcccactttgtggttctaagtactgtggtttccaaatgtgtcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagttctaattccatcagaagcTACTCATCAAGCGTTGCACAGCGTTGCCCCCGAAGCTCCCAGTGA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
23994 | CAG CCT CTG TTC CAC ATA CAC T | Mutant Forward | A | |||
39085 | Fluorophore-1 | GCT GTG CAA CGC TTG ATG | Quencher-1 | Common | A | |
39086 | TCT CTC CAC AGA GCG AGT CA | Wild type Forward | A | |||
39087 | Fluorophore-2 | TTG CCA AGT TCT AAT TCC ATC AGA AG | Quencher-2 | MUT Probe | ||
39088 | Fluorophore-3 | AGA CCT CAT GTT TGG CTA CCA GTT C | Quencher-3 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
23994 | 0.40 uM |
39085 | 0.40 uM |
39086 | 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 |