Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 111 bp
Wild Type = 93 bp
>chr6:114304291+114304383 93bp GTGGCTGCTCACCATCACAT CTCTGTAGCTGCCCCCTCAC
Mutant Sequence:
CCCTGCCATGGAAACAGTGTGACAACCCGTGGAACACTGACCGCTGCTTCTCCAACTACAGCCTGGTCAATACCACCAACATGACCAGCGCCGTGGTGGAGTTCTGGGAGTGAGTGGGGGCTGGGGTGAGGCTGGGGAGGGTGTGCCAGCTGGGTGGTGGCAGGCAATGTGGTGTTTCCTTCAGCCATTCATTCCTGAATGTTTCTTGAATGCCTACTGTGTGCCGGTAGAGATCTGGGATTGCCAATAGACAGTGACAGCAGCAGGTGGCTCATATAGGCAGGGAGACGTGCAGGGTAGGGGAGGTGGCCTGGAACAGCCTACAGGAGAGTGTGGCTGCTCACCATCACATGTGGGGTTGGGTGGTCAGTGGTACTATAACTTCGTATAGCATACATTATACGAAGTTATATACTCTGTATCCTGTTTTCAGTTCATAGCCCATCTGTAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTGGGGATCGACGGTATC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
59439 | Fluorophore-1 | TG+ TA+ T+G +CT ATA +C+ GAA G | Quencher-1 | MUT Probe | ||
60258 | GTG GCT GCT CAC CAT CAC AT | Common | A | |||
60259 | CTC TGT AGC TGC CCC CTC AC | Wild type Reverse | A | |||
60260 | Fluorophore-2 | TCA GTG GTA CTT AGT GGG GAG | Quencher-2 | WT Probe | ||
60261 | GGG CTA TGA ACT GAA AAC AGG | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
60258 | 0.40 uM |
60259 | 0.40 uM |
60261 | 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 |