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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:94972095-94972234 140bp CGTGGGTACCAAGTCATCG CACTTGGGCTGTATGCAAAT
Mutant= 102 bp
Wild Type = 140 bp
Wt Sequence: CGTGGGTACCAAGTCATCGAGgtgccagcatagaggcatcaaggccctgggggtggccactggggcctaagggtaaccctgccagcgggattGaccgtttagtgtgcttatttgcatataatttgcatacagcccaagtg
Mutant Sequence: ctaaagcgcatgctccagactgccttgggaaaagcgcctcccctacccggtagaattgacctgcaggggccctcgatatcaagcttggctggacgtaaactc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
32181 | GAG TTT ACG TCC AGC CAA GC | Mutant Reverse | A | |||
55000 | CAC TTG GGC TGT ATG CAA AT | Wild type Forward | A | |||
55001 | CGT GGG TAC CAA GTC ATC G | Wild type Forward | A | |||
55004 | Fluorophore-1 | CTG CCA GCG GGA TTG A | Quencher-1 | WT Probe | ||
55285 | Fluorophore-2 | CCC TAC CCG GTA GAA TTG ACC T | Quencher-2 | MUT Probe | ||
oIMR5316 | CTA AAG CGC ATG CTC CAG AC | Mutant Forward | A |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
32181 | 0.40 uM |
55000 | 0.40 uM |
55001 | 0.40 uM |
oIMR5316 | 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 |