Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:79834117-79834216 100bp CAACACCATCAGGGATTTGG GAATGCATCCTCTAATACTCACTG
Mut = 91 bp
Wt = 100 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTAGTGACAACATGCAACACCATCAGGGATTTGGCCCTGGGAACCCTCAAGagcgtaaggcaatggttgtgagaatcttagaaggaaagccagtgagtattagaggatgcattctagagttgtgttgaggcagaagttaaattcaggaaaaatggtaatttaactaagaaatgcgtttgttgtgtgggaagtcatccatttttccttcatgttgtatctacaggcaacattgctgtcgttccaattatcaaaaccatcggtttaggacttggaatcctaatctggggatcatttaacaccttaactggctgggcaagctcaaggtaacacagtccacactatctcagctacgagtcctgcatcctgcatttggtgtaaactgggatctagctatattgggagaaataacatgattcttggtctcagagtatggctggtacatgtaaatgaaccatgtctattatttgtctgaagctcttcataagtgatggagatattttatgaagatatagtaaaggaaataaaaagtaaaagctgtatctgggagacaagTGGGGAGAAAGGCAGCTCATGTCTGCTTTCTCCCTCTACTGCCTAACCAGGTACCTAAAGTAGATGCTCGACAATCTGTGGCTCGATCTCCCTGGGATTTTGTAAGATTTAGTGAGATGTAGCTATGTCAAAACACT
This mutation is a 501 bp deletion beginning at Chromosome 3 position 79,833,679 bp and ending after 79,834,179 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50927 | CAA CAC CAT CAG GGA TTT GG | Common | A | |||
50928 | GAA TGC ATC CTC TAA TAC TCA CTG | Wild type Reverse | A | |||
50929 | GTA CCT GGT TAG GCA GTA GAG G | Mutant Reverse | A | |||
50930 | Fluorophore-1 | AGG CAA TGG TTG TGA GAA TCT TAG | Quencher-1 | WT Probe | ||
50931 | Fluorophore-2 | CAA GTG GGG AGA AAG GCA G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50927 | 0.40 uM |
50928 | 0.40 uM |
50929 | 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 |