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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 104 bp
Wild Type = 104 bp
>chr1:64733146-64733249 104bp TACCAGTGACCAAGGCAGAG TGCCTAGACTCCAGCTGTTTC
Wt Sequence:CATTACTGGGAAGGAAATGGCAGGGCGTACCAGTGACCAAGGCAGAGCGGGGACTGAATTTAGGGATCTAAAATGGCCAAGCCACTGAAATGTCACCAAGCTTTCCTAGAGAAACAGCTGGAGTCTAGGCATCCCGATTTTCTTTTGCAGCAGCTGGGGGGTGGGTTGGGGGTAGGGAGAGT
Mutant Sequence: tctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctgttaacttaattaacggccGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCgtcgaccatggatatctgcagatctAAGCTTTCCTAGAGAAACAGCTGGAGTCTAGGCATCCCGATTTTCTTTTGCAGCAGCTGGGGGGTGGGTTGGGGGTAGGGAGAGTTAAGCCTGAGCCA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
24799 | AAT TAA CGG CCG AAG TTC CT | Mutant Forward | A | Mut F | ||
49568 | TAC CAG TGA CCA AGG CAG AG | Wild type Forward | A | Wt F | ||
49569 | TGC CTA GAC TCC AGC TGT TTC | Common | A | |||
49570 | Fluorophore-1 | CGT CGA CCA TGG ATA TCT GCA G | Quencher-1 | MUT Probe | ||
49571 | Fluorophore-2 | ATG GCC AAG CCA CTG AAA TGT C | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
24799 | 0.40 uM |
49568 | 0.40 uM |
49569 | 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 |