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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 136 bp
Wild Type = 149 bp
This assay can be run as a standard assay. Run separated and omit probes.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
38353 | TTA AAA TGA GTG CCC CGA AG | Wild type Forward | A | mChr18 | ||
38354 | CTC CTC TAC CCT AGA TGG TCA A | Common | A | mChr18 | ||
38355 | Fluorophore-1 | CAT TCA CTT AAC TCA CTT GGG GC | Quencher-1 | WT Probe | mChr18 | |
38356 | TTT TCA CTG CAT TCT AGT TGT GG | Mutant Forward | A | SV40pA | ||
38357 | Fluorophore-2 | TTT GTC CAA ACT CAT CAA TGT ATC TTA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
38353 | 0.40 uM |
38354 | 0.40 uM |
38356 | 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 |