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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 77 bp
Wild Type = 96 bp
Wt Sequence:
actttctaaagaatgttcttcccttgtcagATTTTAAATAACCTCAGCATGGTCATTAAGCCAGGGGAAACGACGGCGTTCGTGGGATccagtGGGGCAGG
GAAGAGCACAGCACTACAGCTCATTCAGAGATTCTATGACCCCTGCGAAGGCATGgtgcgtatcttctagaaacttctcaacatggaggggtaagg
Mutant Sequence:
TTTAAATAACCTCAGCATGGTCATTAAGCCAGGGGAAACGACGGCGTTCGTGGGATCCcccgatctatagatcatgagtgggaggaatgagctggcc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
29884 | TCA TTA AGC CAG GGG AAA CG | Common | A | |||
29885 | CGC AGG GGT CAT AGA ATC TC | Wild type Reverse | A | |||
29887 | Fluorophore-1 | TGA GCT GTA GTG CTG TGC TCT | Quencher-1 | WT Probe | ||
39907 | Fluorophore-2 | CGT GGG ATC CCC CGA TCT ATA G | Quencher-2 | MUT Probe | ||
oIMR1653 | GGC CAG CTC ATT CCT CCC ACT CAT G | Mutant Reverse | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
29884 | 0.40 uM |
29885 | 0.40 uM |
oIMR1653 | 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 |