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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:17915993-17916143 151bp GGTGTTTGTTCTAGGGGAGGT CTCCCAAAGCTGCAGATCA
Mut = 131 bp
Wt = 151 bp
Fam = Mut
Hex = Wt
MUT sequence:
ggtgtttgttctaggggaggtgtaactcgcacgaatgggccaggcggacagatggacgcccTCttttgtcaccaagttaacttagagaattaaaggctgctttgtgggtacttggcgtgtgaggtatgagg
This mutation is a 8090 bp deletion beginning at Chromosome 10 position 17,907,992 bp and ending after 17,916,081 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
48611 | GGT GTT TGT TCT AGG GGA GGT | Common | A | |||
48612 | CTC CCA AAG CTG CAG ATC A | Wild type Reverse | A | |||
48613 | CCT CAT ACC TCA CAC GCC AAG | Mutant Reverse | A | |||
48614 | Fluorophore-1 | CTG AAT CTC GTT TTC TTC CTC TTT C | Quencher-1 | WT Probe | ||
48615 | Fluorophore-2 | CCC TCT TTT GTC ACC AAG TTA ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
48611 | 0.40 uM |
48612 | 0.40 uM |
48613 | 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 |