Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr8:70229613-70229732 120bp GGCTGGGTAATACTGGGAAGA GCAGAGAGGAAGATGGGACA
Mut = 120 bp
Wt = 120 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
TATTCAGAGACAGCATCTGTGGCATGGCCTTGCTTGTAGCTTGTGGCTGGGTAATACTGGGAAGAACCctggaaggaaggcccccagcagggacctcctgcctgggggacttgaggaaggatagttgtcacttgactccagcactgtcccatcttcctctctgcaggggttgatctgagcaacattgtgaagacaataccgaaagtatcactggatggccttcaggagcccacacatagtgtccttcaggtaggagcctccgtctccaccaccTTGGTGGAACAGACTGCAGGGGTGGGTGTCTCTTCCCTGTGTTCCATATGGCAGAAGT
This mutation is a 205 bp deletion beginning at Chromosome 8 position 70,229,504 bp and ending after 70,229,708 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49730 | GGC TGG GTA ATA CTG GGA AGA | Common | A | |||
49731 | GCA GAG AGG AAG ATG GGA CA | Wild type Reverse | A | |||
49732 | GGT CAC AAG CTG GAC AGA CC | Mutant Reverse | A | |||
49734 | Fluorophore-1 | CCT TGG TGG AAC AGA CTG CAG | Quencher-1 | MUT Probe | ||
51930 | Fluorophore-2 | AGC AGG GAC CTC CTG CCT G | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49730 | 0.40 uM |
49731 | 0.40 uM |
49732 | 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 |