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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 71 bp
Wild Type = 159 bp
>chr19:11554446+11554604 159bp GAAATATGCAGGAGGGACCTG CAGGTGATTCTCAAGGACACAA
MUT Sequence (junction in uppercase):
tatcttatctaagctattggtttattattattattattattattattattattatttatcacatgggaagatttcatttGGaggcaagggctcactgactcttgcaaaagcaacagtcaacattctgatcacaaaatcaatgttggggtgatacccaaggaggggct
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
53411 | CAG GTG ATT CTC AAG GAC ACA A | Wild type Reverse | A | |||
53414 | Fluorophore-1 | AGG CTC TCC AGT TAA TGT GTC AAA | Quencher-1 | WT Probe | ||
54093 | GAA ATA TGC AGG AGG GAC CTG | Wild type Forward | A | |||
54283 | CAT GGG AAG ATT TCA TTT GG | Mutant Forward | A | |||
54284 | GTG ATC AGA ATG TTG ACT GTT GC | Mutant Reverse | A | |||
54285 | Fluorophore-2 | CAA GGG CTC ACT GAC TCT TGC A | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
53411 | 0.40 uM |
54093 | 0.40 uM |
54283 | 0.40 uM |
54284 | 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 |