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Mutant = gctAGTC
Wild type =atgAGTC
TCCAGCCTGCGAGGCCATCACTTCCTGGTGTCTCTTTGG
GCCAGAGCTTCCAGAAGACCCCCGCAGTGTCCCTGCTAC
CAAAGGGTTGTTGTCCCCGCTC(atg/gtc)AGTCGGCCAGA
GATCAAGGCTGGCGACAGCTCCGGGACAGGAGCAGGAC
AGAAGGTGCTGCCCAAAGGACTGTCACCACCCAGGCAGC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
17454 | GCC ATC ACT TCC TGG TGT CT | Forward | A | |||
17455 | TGG GTG GTG ACA GTC CTT TG | Reverse | A | |||
17456 | Fluorophore-1 | CCC GCT CAT GAG TCG GC | Quencher-1 | WT Probe | ||
17591 | Fluorophore-2 | CCC GCT CGC TAG TCG GC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
17454 | 0.40 uM |
17455 | 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 |