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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr13:52508194-52508288 95bp GCTGGAGTTCTGAAGCAACA TCAAAACGAGGGAGCTCTTG
Mutant= 99 bp
Wild Type = 95 bp
Wt Sequence: GCTGGAGTTCTGAAGCAACATGCCGGAACAGAGCAACGAcTACCGGGTGGCGGTGTTTGGGGCAGGCGGTGTTGGCAAGAGCTCCCTCGTTTTGA
Mut Sequence:
GCTGGAGTTCTGAAGCAACATGCCGGAACAGAGCAACGActGAGAGGCCTTCGTGCAGGAGCTGCTGCTCTCGGGCCTCTCTCTTCCTCCCCTCTGTGA
A 576 bp deletion beginning at Chromosome 13 position 52,507,673 bp and ending after 52,508,248 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
65466 | GCT GGA GTT CTG AAG CAA CA | Common | A | |||
65467 | TCA AAA CGA GGG AGC TCT TG | Wild type Reverse | A | |||
65468 | TCA CAG AGG GGA GGA AGA GA | Mutant Reverse | A | |||
65469 | Fluorophore-1 | AGC AAC GAC TGA GAG GCC TT | Quencher-1 | MUT Probe | ||
65470 | Fluorophore-2 | CGG GTG GCG GTG TTT | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
65466 | 0.40 uM |
65467 | 0.40 uM |
65468 | 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 |