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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 96 bp
>chr9:33514651+33514760 110bp TGGGATATGTTGTACAAAAAGAACT TTAGCTAGCTGAACTCTAAATGAGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40203 | TGG GAT ATG TTG TAC AAA AAG AAC T | Common | A | mChr9 | ||
40204 | TTA GCT AGC TGA ACT CTA AAT GAG C | Wild type Reverse | A | mChr9 | ||
40205 | Fluorophore-1 | ATC AGC ATG CTA GAA TTC CAG TAC ATT | Quencher-1 | WT Probe | ||
40206 | CAC GGG CAA CAC CTT TCC | Mutant Reverse | A | rat | ||
40207 | Fluorophore-2 | ATG TGA CCT GCA GGT CAA CG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40203 | 0.40 uM |
40204 | 0.40 uM |
40206 | 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 |