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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 70 bp
Wild Type = 72 bp
chr4:152332058+152332129 72bp GTCTTTCTCTAGTGGTCTCTCCAG GCTTGGCAATAAAAGTGAGACC
Wt Sequence: gaggtggaaggtgtgtggagccgtcggctcacctgtgcggtctttctctagtggtctctccagaccattctctaGACgtctgctgatatggtctcacttttattgccaagctagggatgtgtgatagacgc
Mutant Sequence: AGACGAGAGGCTGGGTGGTGGGCTCCGGCCAGCCCGAGTCTTAGAGCTGGTGGTTGGTTATATCTGGTGCCTGTCTCGAGGccgctctagtcgacccgtaccttgtcgagtccgataacttcgtataatgtatgctatacgaagttatggcgcgaattcaacgaagttcctatactttctagagaataggaacttcggaataggaacttcgttggatctcgagtttaaacttaagtatcgCTGCTGATATGGTCTCACT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
44837 | GTC TTT CTC TAG TGG TCT CTC CAG | Wild type Forward | A | |||
44838 | GCT TGG CAA TAA AAG TGA GAC C | Wild type Reverse | A | |||
44840 | Fluorophore-1 | CCA TTC TCT AGA CGT CTG CTG A | Quencher-1 | WT Probe | ||
57060 | GCT GGT GGT TGG TTA TAT CTG G | Mutant Forward | A | |||
57061 | TCG GAC TCG ACA AGG TAC G | Mutant Reverse | A | |||
57062 | Fluorophore-2 | CTC GAG GCC GCT CTA GTC GAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
44837 | 0.40 uM |
44838 | 0.40 uM |
57060 | 0.40 uM |
57061 | 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 |