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Mutant Tm = 85C +/- 1
Heterozygote Tm = Mutant 85C +/- 1 + Wild Type 80C +/-1
Wild type Tm = 80C +/- 1
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
12139 | TCA GAA GTT CAG GGC CAC TC | Common | ||||
12140 | GTC CCT ACC TTG TCC CAA AA | Wild type Reverse | ||||
oIMR7572 | CAG ACT GCC TTG GGA AAA GT | Mutant Reverse |
Component | Final Concentration |
---|---|
ddH2O | |
KAPA SYBR Fast | 1.00 X |
12139 | 1.00 uM |
12140 | 1.00 uM |
oIMR7572 | 1.00 uM |
DNA | 0.00 |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 66.0 | -- | . |
4 | -- | repeat steps 2-3 33 times | |
5 | 72.0 | -- | |
6 | 10.0 | -- | hold |