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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:83513349-83513466 118bp TAATTCGGGGCAGCATTAAC GCTGAGCAGTGATTCAGTTCA
Mut = 120 bp
Wt = 118 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
gaccccacccacgtcctgggcccgcccccgctctaggccccgcccagaactcgctctgaccgactgtgtcgctctcagccgcCCataggggactgagcttccctaaacctgagcgttgatttctttttaattagccaccaagccttttttttttttaattta
This mutation is a 12,878 bp deletion beginning at Chromosome 17 position 83,501,735 bp and ending after 83,514,612 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
51557 | TAA TTC GGG GCA GCA TTA AC | Wild type Forward | A | |||
51558 | GCT GAG CAG TGA TTC AGT TCA | Wild type Reverse | A | |||
51559 | GAC CCC ACC CAC GTC CTG | Mutant Forward | A | |||
51560 | ATC AAC GCT CAG GTT TAG GG | Mutant Reverse | A | |||
51561 | Fluorophore-1 | CGA GTG AGC AGA TTT TCT GGC A | Quencher-1 | WT Probe | ||
52912 | Fluorophore-2 | AGA ACT CGC TCT GAC CGA CTG TG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
51557 | 0.40 uM |
51558 | 0.40 uM |
51559 | 0.40 uM |
51560 | 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 |