Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:127192598-127192705 108bp GATGCTGGTCTCCAAGGATG TGATCTCTTACCCCCTGGAC
Mutant= 109 bp
Wild Type = 108 bp
Wt Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG
Deleted Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
37338 | GAT GCT GGT CTC CAA GGA TG | Wild type Forward | A | |||
37339 | TGA TCT CTT ACC CCC TGG AC | Common | A | |||
37340 | GGT TCA GAA GGA GCC TGA AG | Mutant Forward | A | |||
37341 | Fluorophore-1 | AGT CCC TTT CAC CCT GGG | Quencher-1 | MUT Probe | ||
37342 | Fluorophore-2 | CTC CTG CTG CCC AGC TAC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
37338 | 0.40 uM |
37339 | 0.40 uM |
37340 | 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 |