Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr12:76239286-76239431 146bp AGTGGGAATTAGATCTTCTGAAAGG TGTTAAAGGCAGACAGGAATCT
Mut= 161 bp
Wt= 146 bp
Fam=Mut
Hex=Wt
Mut Sequence
gttctgatcaccgggggttcatgaacatacacagacactcaggcatacacatataaacacataaataaataaatatgtaaaaagagaacctggaaccgctcagcccagacccagacctccagtaacactaggacactcaaggaccttggtgttgacccttaagactgaaaggaaagtaatatggggttgcatttatactttcttctggggtataaagagctctctaaactattgtacgcacattatcaatttacatttgaatttttgtggtttggttgtttcttaagtactatagatagcatttatgatagttgaaaacttttgaatgaGGagtttagattcctgtctgcctttaacactcctactgaagatggtgcctcattcagtgcttttagaagtcagaatattgctaacggaataccatgctcaaaatttatgacacaggggccagagagttggctctgcagttaagagctttgagtgtgcttccagaggaccaaggtttaattcctaacatccacatggtagctctgcaactccagttccagaggatctgatgccctgttctgctctctgcaggcatcaggcacacagacatagatgcagaccaaacacccacatgcataaaatttttaaaaagttaaagtaaaaaaatattgtggcataataataactataagcat
This mutation is a 6247 bp deletion beginning at Chromosome 12 position 76,239,314 bp and ending after 76,245,560 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
52516 | AGT GGG AAT TAG ATC TTC TGA AAG G | Wild type Forward | A | |||
52517 | TGT TAA AGG CAG ACA GGA ATC T | Common | A | |||
52518 | CTT TCT TCT GGG GTA TAA AGA GC | Mutant Forward | A | |||
52519 | Fluorophore-1 | AGG GAG TTC TGC TAA CGG TTA G | Quencher-1 | |||
52520 | Fluorophore-2 | ATA GTT GAA AAC TTT TGA ATG AGG AGT | Quencher-2 |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
52516 | 0.40 uM |
52517 | 0.40 uM |
52518 | 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 |