Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr4:152117892+152117993 102bp TCCCTTCCACTCCCATATTTAG GCCAAGGGCTAGCTGCAAAG
Mut= 123 bp
Wt= 102 bp
Fam=Mut
Hex=Wt
Wt Sequence:
ttacgcggatacataaaagagcagatattatccctatcccttccactcccatatttagagtagacttaccgtttatttgcttacactgatagagctttgaaacttcagctaagactggctttgcagctagcccttggcaagttgaaagatttcaggtttaccctctgaacgtgggactg
Mutant Sequence:
AGCAGAACGGTAAAGGGTTACGCGGATACATAAAAGAGCAGATATTATCCCTATCCCTTCCACTCCCATATTTAGAGTAGACTTACCGTTTATTTGCTTACACTGATAGAGCTTTGAAACTTCAGGCCGGCCCAATTGTTCGAAGGCGCGCCATTTAAATGAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGGTTACCATAACTTCGTATAGCATACATTATACGAAGTTATCCTAGGCTCGAGGCTAAGACTGGCTTTGCAGCTAGCCCTTGGCAAGTTGAAAGATTTCAGGTTTACCCTCTGAACGTGGGACTGCAATAAGGGGAAAGGATACCCAAGCCCCTGAGTATCCCCTGCTTCCCAGACACTGACTCTAGGTGCGAGGACCACTACCCA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51986 | TCC CTT CCA CTC CCA TAT TTA G | Wild type Forward | A | |||
51987 | GCC AAG GGC TAG CTG CAA AG | Common | A | |||
51988 | GAA GAA TAG GAA CTT CGG AAT AGG | Mutant Forward | A | |||
51989 | Fluorophore-1 | AGC TTT GAA ACT TCA GCT AAG ACT GG | Quencher-1 | WT Probe | ||
51990 | Fluorophore-2 | ACG AAG TTA TCC TAG GCT CGA GG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51986 | 0.40 uM |
51987 | 0.40 uM |
51988 | 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 |