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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:48348428+48348522 95bp TTCCAAGCAAGTGGCTGTCT ATGACACCCTCAAGGACTGG
Mutant= 104 bp
Wild Type = 95 bp
Wt Sequence: ttccaagcaagtggctgtctcctctgcagCCCTGTCccaagccctcaacaccacggtgctgcagggcatggccggccagtccttgagggtgtcatgtacttatgacgccttgaagcactg
Mutant Sequence: ttccaagcaagtggctgtctcctctgcagCCCTGTCCTGAAGAAGCGGAATGGGAGCACAGTCATCGCAGATGACACCCTTGCTGGAACCGTCACCATCACTCT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
21122 | ATG ACA CCC TCA AGG ACT GG | Wild type Reverse | A | |||
24643 | TTC CAA GCA AGT GGC TGT CT | Common | A | |||
55471 | AGA GTG ATG GTG ACG GTT CC | Mutant Reverse | A | |||
55473 | Fluorophore-1 | TGC AGC CCT GTC CTG AA | Quencher-1 | MUT Probe | ||
56122 | Fluorophore-2 | CTC AAC ACC ACG GTG CTG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
21122 | 0.40 uM |
24643 | 0.40 uM |
55471 | 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 |