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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:87780057-87780161 105bp CTTGGGGACCAGTGGGTATC CCTAACTCACACGGGCAGT
Mut= 120 bp
Wt= 105 bp
Fam=Mut
Hex=Wt
Mut Sequence:
cctgggaattgcctctaatctctcctctgccttcccctgcagATGGTGATGACGACCCACAGCTCTCCTGGgtaggtcttatcgcaacccagagaggcagcactgcccgtgtgagttagg
This mutation is a 3279 bp deletion beginning at Chromosome 8 position 87,780,106 bp and ending after 87,783,384 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45658 | CTT GGG GAC CAG TGG GTA TC | Wild type Forward | A | |||
45659 | CCT AAC TCA CAC GGG CAG T | Common | A | |||
45661 | Fluorophore-1 | AAC TCT TGC ATG TGT GCT TCC | Quencher-1 | WT Probe | ||
47218 | CCT GGG AAT TGC CTC TAA TCT | Mutant Forward | A | |||
47219 | Fluorophore-2 | CAC AGC TCT CCT GGG TAG GTC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45658 | 0.40 uM |
45659 | 0.40 uM |
47218 | 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 |