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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:121106770-121106869 100bp GCAGGTAACACCACCTCCTG CTCACTGGATGGGAAATTGG
Mutant= 109 bp
Wild Type = 100 bp
Wt Sequence: GCAGgtaacaccacctcctggagggaggatgggcttgctgggaactgttagctaacaaagggcActgttcgaggtgctggccaatttcccatccagtgag
Mut Sequence: ttcccaaaagtctggagttatctaataattttcatagtctatattttttatagcatagattttgttgctagCActgttcgaggtgctggccaatttcccatccagtgag
A 417 bp deletion beginning at Chromosome 2 position 121,106,807 bp and ending after 121,107,223 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66154 | Fluorophore-1 | CTT GCT GGG AAC TGT TAG CTA AC | Quencher-1 | WT Probe | ||
66155 | GCA GGT AAC ACC ACC TCC TG | Wild type Forward | A | |||
66156 | CTC ACT GGA TGG GAA ATT GG | Common | A | |||
66157 | TTC CCA AAA GTC TGG AGT TAT C | Mutant Forward | A | |||
66158 | Fluorophore-2 | TGT TGC TAG CAC TGT TCG AGG T | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66155 | 0.40 uM |
66156 | 0.40 uM |
66157 | 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 |