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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 100 bp
Wild Type = 98 bp
chr3:133471397-133471494 98bp ACACATCTGTCTGTCAGGTTGA CAAACATGCAGTGACTCCTGA
Wt Sequence: ttgttcaacagtagcaatttgaaattagatggtgtcagagggaatttaggtcatgccactttagaagcctattggaaacatttctTTGTGAGCatgggatagtggggatgtaacagtatttccattttttaaacaaactgctcagcattgaatgtagttagtccagagaattttatccacacttaaaatgtccagt
Mutant Sequence: ATTGTTCAACAGTAGCAATTTGAAATTAGATGGTGTCAGAGGGAATTTAGGTCATGCCACTTTAGAAGCCTATTGGAAACATTTCTggatatcgcggccgcatAACTTCGTATAATGTATGCTATACGAAGTTATtaggtggatccgaagcttatcgataccgtcgacctcgacAATAACCCTTGCTGTATGTCTATAATTAGAGCTACTAAAAAGAAATCCAGTGTTTCCTTAAGCAGTCCTCTTTGCTCTGAGAGGTAAAAGTCCCCTCCTTGCTGGTTTCCATGATAACACTGTTCATTTCC
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
54641 | AGG TCA TGC CAC TTT AGA AGC | Mutant Forward | A | |||
54642 | TCG GAT CCA CCT AAT AAC TTC G | Wild type Reverse | A | |||
54643 | ACA CAT CTG TCT GTC AGG TTG A | Wild type Forward | A | |||
54645 | CAA ACA TGC AGT GAC TCC TGA | Wild type Reverse | A | |||
54646 | Fluorophore-1 | CTG GAT ATC GCG GCC GC | Quencher-1 | MUT Probe | ||
54648 | Fluorophore-2 | CAC CAA GCC CCA GAC TGC TG | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
54641 | 0.40 uM |
54642 | 0.40 uM |
54643 | 0.40 uM |
54645 | 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 |