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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 117 bp
Wild Type = 126 bp
>chr6:113025997-113026122 126bp GAGTTCTCTGCTGCCTCCTG AGCCTGCCCAGAAGACTCC
Wt Sequence: caatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctTTCTagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaatag
Mutant Sequence: AAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGatcgccgtgactagaggcaataaaaagacagaataagacgcacgggtgttgggtcgtttgttcgaattcaCTAGAAGATGGGCGGGAGTCTTCTGGGCAGGCTTA
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
24493 | AGC CTG CCC AGA AGA CTC C | Common | A | |||
33533 | Fluorophore-1 | CAA CTC CAG TCT TTC TAG AAG ATG G | Quencher-1 | WT Probe | ||
39497 | AGA AGG GCG GAA AGA TCG | Mutant Forward | A | |||
39499 | Fluorophore-2 | ACG CAC GGG TGT TGG GTC | Quencher-2 | MUT Probe | ||
oIMR4981 | GAG TTC TCT GCT GCC TCC TG | Wild type Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
24493 | 0.40 uM |
39497 | 0.40 uM |
oIMR4981 | 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 |