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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr14:87487413+87487525 113bp CATGTCGCCAAAACATAGGA GGTTGGGTGGAGTACTTTGG
Mut = 137 bp
Wt = 108 bp
Fam = Mut
Hex = Wt
MUT Sequence (junction in uppercase):
ttatgatgtctttatatcatctcattttcatctgcattgatacaCAtggaaagttagatgtacaaacagatagttgagttggaaaatttactgtagcatgacttcatattgcttacatcatttctgagattggtctgtctgttttcctcacatgt
This mutation is a 2230 bp deletion beginning at Chromosome 14 position 87,485,304 bp and ending after 87,487,533 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50434 | CAT GTC GCC AAA ACA TAG GA | Wild type Forward | A | |||
50437 | GGT TGG GTG GAG TAC TTT GG | Wild type Reverse | A | |||
50440 | Fluorophore-1 | TGC ATT CTG AGA ACA AAG GCC | Quencher-1 | WT Probe | ||
51756 | TCA TCT CAT TTT CAT CTG CAT TG | Mutant Forward | A | |||
51757 | ATG TGA GGA AAA CAG ACA GAC CA | Mutant Reverse | A | |||
51758 | Fluorophore-2 | CTG TAG CAT GAC TTC ATA TTG CTT ACA TCA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
50434 | 0.40 uM |
50437 | 0.40 uM |
51756 | 0.40 uM |
51757 | 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 |