Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 100 bp
Wild Type = 101 bp
>chr2:180283515-180283615 101bp AGCCACTTTTAGAACCAACCTCT GGACACCCCATCTACACCTC
Wt Sequence AGACAGGTCCAGGAGAAGTTAAGGGTGTATGTAGCTTCCCTTCCCTACCCCTCAAAAAGTAGCCCCTCTTGGATACTCTGCCCCTCCAGATGGCCAGCCACTTTTAGAACCAACCTCTAAtcagatgctaagctagagtcccaggccacagctcaggaaaccaggacatctgggcagaggtgtagatggggtgtcctctccacacctttcccagggtctgcttccaagagtcaggcggatatcaggggtctggagatgctaaccagggcaagagtttccatggcccgcctcagccctaccccggctgtgagggggcagctgggcggggcctgacttgcactttctctgtcctctctgccagGTCTGCAGAACAAGCTTCTAGAACTGAACTCAGAACGGTGCCGgtgagtgagcccagcccagccagaggagcagatgggagggcagctatccccacaagccatagtcaccaacactgcccatgaggtcccaggatggcagctcacccagtgtcccaaatgaaGAAGGGAAACTGAGGTTTAGCATGGCAGGGAGGGGGCAGCGAGTCCCTTGGCCAAGGTGACATACTACTCAAGTGACAGACCATTGAGACATCAGTCTCAAGCGGTCTGACATGGCCCTCCACTTGGGAACCAA
Mut Sequence ggagaagttaagggtgtatgtagcttcccttccctacccctcaaaaagtagcccctcttggatactctgcccctccagatggccagccacttttagaaccaacctctaAGaagggaaactgaggtttagcatggcagggagggggcagcgagtcccttggccaaggtgacatactactcaagtgacagaccattgagacatcagtctcaagcggtctgacatggccctccacttgggaacc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44413 | CAC TTG AGT AGT ATG TCA CCT TGG | Mutant Reverse | A | |||
44414 | AGC CAC TTT TAG AAC CAA CCT CT | Common | A | |||
44415 | GGA CAC CCC ATC TAC ACC TC | Wild type Reverse | A | |||
47211 | Fluorophore-1 | AGC TCA GGA AAC CAG GAC ATC | Quencher-1 | WT Probe | ||
47213 | Fluorophore-2 | AGG TTT AGC ATG GCA GGG AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44413 | 0.40 uM |
44414 | 0.40 uM |
44415 | 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 |