Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Wt Sequence: cagcttcagcctgaagagtaagtagttctctattggcagtttgacacatcctgcccttaccttactaccactgttggctcagcagacacccaggataagtgcactagtgttcctttcctgaccagtgggactgctttttccagATTCCtcttgaagctgaggaggcagtgagagcatgatggcatctaatgagcttggaagtaccagactgccctgatccacagccaggttttgctgaaaagtgaccagtttgtcctcctccagtagagtgggcagctgaagGATTATAatctactgtcaagacttggaggcccctgcagtcaaagtccaatagaatattatgaaatggagaatggcttattttaatctctatagtggaatt
Mutant Sequence: CAGCAGACACCCAGGATAAGTGCACTAGTGTTCCTTTCCTGACCAGTGGGACTGCTTTTTCCAGATTCCTCTTGAAGCTGAGGAGGCAGTGAGAGCATGATGGgcgcgccccttactaggcggccgcgaagttcctataccttttgaagaataggaacttcggaataggaacttcgtcgacaaaggatccgtcggcgaccctacgcccccaactgagagaactcaaaggttaccccagttggggcactactcccgaaaacgtcggcgaccctacgcccccaactgagagaactcaagggcacgccctggcacccgcaccgcggcttcgagaccgtgacctac
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52350 | AGG AGG CAG TGA GAG CAT GA | Common | A | Igs2 | ||
52351 | TCT TCA AAA GGT ATA GGA ACT TCG | Mutant Reverse | A | Vector | ||
52352 | AGC AAA ACC TGG CTG TGG | Wild type Reverse | A | Igs2 | ||
52353 | Fluorophore-1 | CGC CCC TTA CTA GGC GGC C | Quencher-1 | MUT Probe | ||
52354 | Fluorophore-2 | AGC TTG GAA GTA CCA GAC TGC CC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52350 | 0.40 uM |
52351 | 0.40 uM |
52352 | 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 |