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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 93 bp
Wild Type = 94 bp
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
This protocol can be used for conventional PCR. Omit probes and run as a separated assay.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
40119 | TAT TAT GAT GAT ATT TGG AGG CTG A | Common | A | mChr14 | ||
40120 | TTT TAA GGG CAC TAT GTG ATC C | Wild type Reverse | A | mChr14 | ||
40121 | Fluorophore-1 | TTT ATG GTG ACA TAG GAG AAG GAT TG | Quencher-1 | WT Probe | ||
40122 | GGT AAC CGG CCT CTT CAT C | Mutant Reverse | A | vector | ||
40123 | Fluorophore-2 | TAT GGT GAT GCT GAA GGT CG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
40119 | 0.40 uM |
40120 | 0.40 uM |
40122 | 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 |