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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 81 bp
Wild Type = 115 bp
>chr16:91492910+91493024 115bp TGTCAACATACTACAACGACCAAG CATTCCACGAAGATGTGCTG
Wt Sequence:AAGAAAAGACGAGGCGAAGTGGTTAAAAGTGCCTGAATGTCAACATACTACAACGACCAAGTGtgaattctCTTTACTGGACACAAATGTGTATATCAAAACACAGTTTCGTGTCAGAGCAGAGGAAGGGAACAGCACATCTTCGTGGAATGAGGTTGATCCGTTTATTCCATTCTACACAGGTAAGAAGCCGCCATGGCTGGCTATAT
Mutant Sequence: AAAAGTGCCTGAATGTCAACATACTACAACGACCAAGTGTGAATTCgataagcttgattcgagcagtgtggttttcaagaggaagcaaaaagcctctccacccaggcctggaatgtttccacccaatgtcgagcagtgtggttttgcaagaggaagcaaaaagcctctccacccaggcctggaatgtttccacccaatgtcgagcaaaccccgcccagcgtcttg
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38760 | TGT CAA CAT ACT ACA ACG ACC AAG | Common | A | |||
38761 | GGC TTT TTG CTT CCT CTT GA | Mutant Reverse | A | |||
38762 | CAT TCC ACG AAG ATG TGC TG | Wild type Reverse | A | |||
38763 | Fluorophore-1 | AAG CTT GAT TCG AGC AGT GTG GTT | Quencher-1 | MUT Probe | ||
38764 | Fluorophore-2 | AAA CAC AGT TTC GTG TCA GAG CAG AG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38760 | 0.40 uM |
38761 | 0.40 uM |
38762 | 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 |