Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 109 bp
Wild Type = 117 bp
>chr6:86367604+86367720 117bp AGGCCAGCCAACACTACAGA ACAGCACCATGGGGAAAAG
Wt Sequence: atggccaggtgaggtatggggcctagccttttaatcaaaacagtcaggaagcagaagaaggcagatttctatgagttcaaggccagccaacactacagagtctagAATGaatgagagaaggaaagaaagaaacaaattaggctaaagcctctagaaactggagacctatgttacctccttttccccatggtgctgtgcacatgctaactaccagatagttatggaactactcaaatttgaatgaaacataacagtaatatggaaaactaatggagaggaacccaggggaaatgagtggataactttaaattgtggctcggagtggtggtgcatgcctttaatccagcactagggaagcagaggcaggtggatc
Mutant Sequence:CAGGGCCAGCTCTTCAGCTTGGCCTGGATGAGGGGCAAGGCCAAATCTCCTGCTTATAGCCAAAAGTCAGGACTGACTCACATGTTCAGGACTGGCTCTCAGGGCCAGCTTGCCCTTCATGGCCAGGTGAGGTATGGGGCCTAGCCTTTTAATCAAAACAGTCAGGAAGCAGAAGAAGGCAGATTTCTATGAGTTCAAGGCCAGCCAACACTACAGAGTCTAGAActagtggatcccccgggctgcaggaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgcta
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40145 | CTA AAG CGC ATG CTC CAG A | Mutant Reverse | A | |||
40147 | Fluorophore-1 | ACC GGG TAG GGG AGG CGC | Quencher-1 | MUT Probe | ||
40181 | AGG CCA GCC AAC ACT ACA GA | Common | A | |||
40182 | ACA GCA CCA TGG GGA AAA G | Wild type Reverse | A | |||
40183 | Fluorophore-2 | AGG CTA AAG CCT CTA GAA ACT GGA GAC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40145 | 0.40 uM |
40181 | 0.40 uM |
40182 | 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 |