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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:41158744+41158852 109bp CAGCAACTGGAAAAAGGATGA CTTGAAGGAAGCTTGACAGGA
Mutant= 108 bp
Wild Type = 109 bp
Wt Sequence: CAGCAACTGGAAAAAGGATGAGCCAAATAACCATGGctCTGGGGAAGACTGTGTCATTATATTAGATAATGGTTTGTGGAATGACATTTCCTgtcaagcttccttCAAG
Mutant Sequence: CAGCAACTGGAAAAAGGATGAGCCAAATAACCATgctagagtcagcttctgatggaattagaacttggcaaaacaatactgagaatgaagtgtatgtggaacagaggc
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
46296 | CAG CAA CTG GAA AAA GGA TGA | Common | A | |||
46297 | CTT GAA GGA AGC TTG ACA GGA | Wild type Reverse | A | |||
46298 | GCC TCT GTT CCA CAT ACA CTT C | Mutant Reverse | A | |||
46299 | Fluorophore-1 | CAT GCT AGA GTC AGC TTC TGA TGG | Quencher-1 | MUT Probe | ||
46300 | Fluorophore-2 | CTC TGG GGA AGA CTG TGT CA | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
46296 | 0.40 uM |
46297 | 0.40 uM |
46298 | 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 |