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Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 92 bp
Wild Type = 92 bp
>chr7:5018426+5018517 92bp CCTCCTGTGGACTGAGAAGC CAGGCACCATGACTGAACAC
Large deletion: Mutant sequence with junction in uppercase
catgtcttactaacttatacagaatgtcttatatggatctcaaaagtctttagaaggaataggtgagatactggggtATcacacatctccctagcttggtgccaaagaaccaatgacttggaacaagcctttgcccaatctgacctccaccagacaagtactacaaaaattttgacctactatgcaaacgtgagtcaaggctccatcctagttaaattcttccaaattctccatagacac
This mutation is a 1202 bp deletion beginning at Chromosome 7 position 5,017,385 bp and ending after 5,018,586 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
66632 | CCT CCT GTG GAC TGA GAA GC | Wild type Forward | A | |||
66633 | CAG GCA CCA TGA CTG AAC AC | Wild type Reverse | A | |||
66634 | Fluorophore-1 | ATC CCT GCC TAC TGA CTT CCA | Quencher-1 | WT Probe | ||
66635 | TTA GAA GGA ATA GGT GAG ATA CTG G | Mutant Forward | A | |||
66636 | AGA TTG GGC AAA GGC TTG TT | Mutant Reverse | A | |||
66637 | Fluorophore-2 | CCC TAG CTT GGT GCC AAA GAA CC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
66632 | 0.40 uM |
66633 | 0.40 uM |
66635 | 0.40 uM |
66636 | 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 |